Use of microproteins as tryptase

ABSTRACT

Disclosed are uses of microproteins preferably microproteins forming a cystine knot (i.e. belonging to the family of inhibitor cystine knot (ICK) polypeptides) or polynucleotides encoding said microproteins for the preparation of a pharmaceutical composition for treating or preventing a disease that can be treated or prevented by inhibiting the activity of tryptase as well as corresponding methods of treatment. Also disclosed are uses of the microproteins for inhibiting tryptase activity, for purifying tryptase, as a carrier molecule for tryptase and for deleting or quantifying tryptase in a sample, including corresponding diagnostic applications. Furthermore disclosed are fusion proteins comprising an inactive barnase as well as fusion proteins comprising barnase and a microprotein. Also encompassed are nucleic acid molecules encoding such a fusion protein, as well as corresponding vectors, host cells, preparation methods and uses of the fusion protein. Moreover, the present application discloses a crystal of a microprotein fused with barnase, preferably inactive barnase. The disclosure also refers to corresponding preparation methods for the crystal, structure analysis methods using the crystal data storage media comprising the structure data obtained, as well as to in silico methods using the structure data for characterizing the binding of microproteins to target molecules. Furthermore, disclosed are pharmaceutical compositions comprising the crystal and corresponding medical uses.

The present application relates to the use of microproteins, preferably microproteins forming a cystine knot (i.e. belonging to the family of inhibitor cystine knot (ICK) polypeptides), or polynucleotides encoding said microproteins for the preparation of a pharmaceutical composition for treating or preventing a disease that can be treated or prevented by inhibiting the activity of tryptase as well as to corresponding methods of treatment. The present invention also relates to uses of the microproteins for inhibiting tryptase activity, for purifying tryptase, as a carrier molecule for tryptase and for detecting or quantifying tryptase in a sample, including corresponding diagnostic applications. The present invention furthermore relates to fusion proteins comprising an inactive barnase as well as to fusion proteins comprising barnase and a microprotein. Also encompassed by the present invention are nucleic acid molecules encoding such a fusion protein, as well as corresponding vectors, host cells, preparation methods and uses of the fusion protein. Moreover, the present invention relates to a crystal of a microprotein fused with barnase, preferably inactive barnase. The present invention also refers to corresponding preparation methods for the crystal, structure analysis methods using the crystal, data storage media comprising the structure data obtained, as well as to in silico methods using the structure data for characterizing the binding of microproteins to target molecules. Furthermore, the invention relates to pharmaceutical compositions comprising the crystal and corresponding medical uses.

Asthma is a complex disease involving multiple biochemical mediators for both its acute and chronic manifestations. Increasingly, asthma is recognized as an inflammatory disorder (see, e.g., Hood, et al., 1984). Asthma is frequently characterized by progressive development of hyperresponsiveness of the trachea and bronchi to both immunospecific allergens and chemical or physical stimuli. The hyperresponsiveness of asthmatic bronchiolar tissue is thought to result from chronic inflammation reactions, which irritate and damage the epithelium lining the airway wall and promote pathological thickening of the underlying tissue. Bronchial biopsy studies have indicated that even patients with mild asthma have features of inflammation in the airway wall.

One initiator of the inflammatory sequence is an allergic response to inhaled allergens. Leukocytes carrying IgE receptors, mast cells and basophils, but also monocytes, macrophages, and eosinophils, are present in the epithelium and underlying smooth muscle tissues of bronchi where they are activated initially by binding of specific inhaled antigens to the IgE receptors. Activated mast cells release a number of preformed or primary chemical mediators of the inflammatory response and enzymes. Furthermore, numerous secondary mediators of inflammation are generated in situ by enzymatic reactions of activated mast cells, including superoxide and lipid derived mediators. In addition, several large molecules are released by degranulation of mast cells: proteoglycans, peroxidase, arylsulfatase B, and notably the proteases tryptase and chymotryptic proteinase (chymase).

This release of compounds from mast cells probably accounts for the early bronchiolar constrictor response that occurs in susceptible individuals after exposure to airborne allergens. The early asthmatic reaction is maximal at around fifteen minutes after allergen exposure; recovery occurs over the ensuing one to two hours. In 25-35% of individuals, the early asthmatic reaction is followed by a further decline in respiratory function which begins within a few hours and is maximal between six and twelve hours post-exposure. This late asthmatic reaction is accompanied by a marked increase in the number of inflammatory cells infiltrating bronchiolar smooth muscle and epithelial tissues, and spilling into the airways. These cells include eosinophils, neutrophils, and lymphocytes, all of which are attracted to the site by release of mast cell derived chemotactic agents. The infiltrating cells themselves become activated during the late reaction phase. The late asthmatic response is believed to be a secondary inflammatory reaction mediated in part by the secretory activity of macrophages.

Human tryptase is a serine proteinase which is the predominant protein present in human mast cells. The term tryptase covers four closely related enzymes (α, I, II/β, III; possessing 90 to 98% sequence identity) (Miller et al. 1989; Vanderslice et al., 1990).

Tryptase is the major secretory protease of human mast cells and is proposed to be involved in neuropeptide processing and tissue inflammation. Mature human tryptase is a tetrameric glycosylated molecule, is heparin-associated and composed of heterogenous, catalytically active subunits (see, e.g., Vanderslice et al., 1990; Miller et al., 1989, 1990, Sommerhoff et al., 1999).

Tryptase is stored in mast cell secretory granules. After mast cell activation, human tryptase can be found in various biologic fluids. Tryptase levels in lung lavage fluid obtained from atopic asthmatics increase after endobronchial allergen challenge. Some smokers of cigarettes have striking elevations of bronchoalveolar lavage fluid tryptase levels compared to nonsmoker control groups, a finding that provides some support for the hypothesis that release of proteinases from activated mast cells could contribute to lung destruction in smoker's emphysema, (Kalenderian, et al., Chest 94:119-123, 1988). In addition, tryptase has been shown to be a potent mitogen for fibroblasts, suggesting its involvement in pulmonary fibrosis and interstitial lung diseases (Ruoss et al., 1991).

Tryptase has been implicated in a variety of biological processes, including degradation of vasodilating and bronchorelaxing neuropepudes (see Caughey, et al., 1988; Franconi, et al., 1989; and Tam, et al. 1990) and modulation of bronchial responsiveness to histamine (see Sekizawa, et al., 1989) and psoriasis. These studies suggest that tryptase possibly increases bronchoconstriction in asthma by destroying bronchodilating peptides.

Elevated levels of mast-cell tryptase have been found

-   -   in the plasma of patients with mastocytosis, after systemic         anaphylaxis (Schwartz et al., 1987, 1989).     -   in the duodenal mucosa of psoriasis patients (Michaelsson et         al., 1997).     -   in bronchoalveolar lavage fluid of patients with asthma (Broide         et al., 1991; Wenzel et al., 1988), interstitial lung diseases         (Walls et al., 1991), and after antigen challenge of allergic         patients (Castells & Schwartz 1988).     -   in the skin blister fluid after cutaneous antigen challenge in         patients with atopic and allergic skin disease (Shalit et al.,         1990; Atkins et al., 1990; Brockow et al., 2002).     -   in nasal lavage fluid after local antigen challenge of patients         with seasonal allergic rhinitis (Juliusson et al., 1991;         Howarth, 1995)     -   in the crevicular fluid of patients with gingivitis and         periodontitis (Cox & Eley, 1989) and in the lesional skin of         patients with psoriasis (Michaelsson et al., 1997).     -   in the mucosa of the ileum and colon of patients with         inflammatory bowel disease (IBD), which was accompanied by great         changes of the content in mast cells such as dramatically         increased expression of TNFalpha, IL-16 and substance P. The         evidence of mast cell degranulation was found in the wall of         intestine from patients with IBD with immunohistochemistry         technique. The highly elevated histamine and tryptase levels         were detected in mucosa of patients with IBD, strongly         suggesting that mast cell degranulation is involved in the         pathogenesis of IBD (He, 2004).     -   in myeloblasts in patients with acute myeloid leukaemia (AML)         that produce significant amounts of tryptase(s). In these         patients, myeloblasts express alpha-tryptase mRNA in excess over         beta-tryptase mRNA, and secrete the respective protein         (=pro-alpha-tryptase) in a constitutive manner (Sperr et al.,         2001, 2002).

Human tryptase is inhibited by small molecular weight substances (e.g. leupeptin and diisopropyl fluorophosphate). Divalent cations, such as calcium, and benzamidine and its derivatives are competitive inhibitors of human mast cell tryptase (Schwartz, 1994). Several low-molecular-weight compounds have been described as tryptase inhibitors in the patent literature (summarized in Newhouse 2002). However, none of the compounds have made their way into later stage clinical trials. This is explained by undesired side reactions, insufficient selectivity, high toxicity, low stability and/or low bioavailability of the different inhibitor compounds described (Newhouse 2002).

Although tryptase has trypsin-like properties, most protein-based inhibitors do not inhibit it. Although having trypsin-like properties, it is a characteristic of human tryptase not to be inhibited by potent trypsin inhibitors such as bovine pancreatic trypsin inhibitor (Di Marco & Priestle, 1997). Endogenous inhibitors that target the catalytic sites of mast cell tryptase have yet to be reported. Human tryptase activity is inhibited by lactoferrin and myeloperoxidase (both neutrophil-derived) and by antithrombin-III, all of which antagonise the glycosaminoglycans (heparin or chondroitin sulfate) that stabilize the mast cell tryptase (MCT) tetramer (Alter et al., 1990; Cregar et al., 1999; Elrod et al., 1997). The only two known protein-based human tryptase inhibitors which inhibit tryptase via tight binding to its active site are the leech derived tryptase Inhibitor (LDTI) and the tick-derived protease inhibitor protein (rTdPI) (WO 95/03333 and WO 01/05832). LDTI is a 46 residue protein, where two LDTI monomers interact with one tryptase tetramer. A recombinant form of this Kazal-type protein has been found to efficiently inhibit 2 of the 4 catalytic sites of the tetrameric tryptase (Stubbs et al., 1997; Auerswald et al., 1994; Sommerhoff et al., 1994) with a Ki of 1.4 nM, while the remaining two sites are inhibited with Ki values of 560 and 10,000 nM, respectively. The efficient binding of only two catalytic sites out of four in the tryptase tetramer has so far prevented any therapeutic uses of LDTI.

Thus, it would be desirable to provide further inhibitors to tryptase, especially for therapeutic purposes. In case of proteinacious inhibitors, it would be furthermore desirable to provide the inhibitor in crystalline form so as to facilitate structure analysis and have a basis for studying and improving tryptase binding. In addition, it would be desirable to provide corresponding means and methods that may be useful for improving the production of such inhibitors or crystals thereof.

In view of the above explanations, it is clear that there is still an on-going need for efficient inhibitors of tryptase. Thus, the technical problem underlying the present invention is to make available further tryptase inhibitors that can be used to prevent or treat diseases that can be prevented or treated by inhibiting tryptase activity. Preferably, such inhibitors should overcome drawbacks associated with tryptase inhibitors of the prior art such as undesired side reactions, insufficient selectivity, high toxicity, low stability, low bioavailability and/or insufficient binding affinity.

This technical problem is solved by the provision of the embodiments as characterized in the claims.

Accordingly, the present invention relates to the use of a microprotein or a polynucleotide encoding said microprotein for the preparation of a pharmaceutical composition for treating or preventing a disease that can be treated or prevented by inhibiting the activity of tryptase.

The present invention is based on the surprising finding that microproteins are capable of efficiently binding tryptase. This is shown for many exemplary specimens in Example 3, infra. Three of the microproteins of the invention were furthermore positively tested for tryptase selectivity (see Table 2 in Example 3, infa). Thus, the use of the present invention refers to the use of microproteins which are capable of significantly inhibiting the activity of tryptase. Preferably, the microproteins are able to bind all four catalytic sites of the tryptase tetramer. The provision of the present invention, i.e. the recognition that microproteins can be used to inhibit tryptase in particular for therapeutic purposes, overcomes disadvantages that are known for low-molecular weight tryptase inhibitors (see, e.g., Newhouse, 2002). For instance, such small molecules may show a toxic effect to the organism to which they are applied due to a relatively low binding specificity causing binding to molecules other than tryptase. Compared to the small molecules, microproteins show a larger interaction surface so that a more selective binding can be expected for them. Furthermore, protein-based binding molecules typically have a lower dissociation rate constant than low-molecular weight molecules, thus, binding for a longer time to the target and therefore having more advantageous binding properties.

In addition, a further advantage over low-molecular weight tryptase inhibitors lies in the fact that microproteins can be expected not to be able to cross the membrane barrier. This prevents microproteins from binding to tryptase stored within mast cells which may potentially influence the physiological state of the mast cell negatively. Small molecules, by contrast, can often cross membranes. Moreover, especially cystine knot proteins are notoriously stable against enzymic or thermal degradation.

The term “microprotein” generally refers to polypeptides with a relatively small size of not more than 50 amino acids and a defined structure based on intra-molecular disulfide bonds. Microproteins are typically highly stable and resistant to heat, pH and proteolytic degradation. The current knowledge on microproteins, in particular in regard to their structure and occurrence, is for instance reviewed in Craik (2001); Pallaghy (1994); and Craik (J. Mol. Biol. 294 (1999), 1327-1336).

In a preferred embodiment, the microprotein in the use of the invention comprises at least six cysteine residues, of which six cysteine residues are connected via disulphide bonds so as to form a cystine knot.

Such microproteins are also known as inhibitor cystine knot (ICK) polypeptides and are also called like that in the following explanations.

The term “cystine knot” refers to a three-dimensional structure formed by the ICK polypeptides which are characterized by a small triple β-sheet which is stabilized by a three-disulfide bond framework which comprises an embedded ring formed by two disulphide bonds and their connecting backbone segments, through which a third disulfide bond is threaded. Preferably, the cystine knot is formed by six conserved cysteine residues and the connecting backbone segments, wherein the first disulfide bond is between the first and the fourth cysteine residue, the second disulfide bond between the second and the fifth cysteine residue and the third disulfide bond between the third and the sixth cysteine residue, the third disulfide bond being threaded through the ring formed by the other two disulfide bonds and their connecting backbone segments. FIG. 11 shows an example of a corresponding cystine knot forming microprotein. If considered suitable, a disulfide bond may be replaced by a chemical equivalent thereof which likewise ensures the formation of the overall topology of a cystine knot. For testing whether a given microprotein has formed the correct cystine knot, a skilled person can determine which cystine residues are connected with one another. This can, for instance, be done according to techniques described in Gorasson (J. Biol. Chem. 278 (2003), 48188-48196) and Horn (J. Biol. Chem. 279 (2004), 35867-35878). Microproteins with a cystine knot are for instance described in Craik (2001); Pallaghy (1994); and Craik (J. Mol. Biol. 294 (1999), 1327-1336).

The microproteins for use in connection with the present invention may have a peptide backbone with an open or a circular conformation. The open conformation preferably refers to microproteins with an amino-group at the N-terminus and a carboxyl-group at the C-terminus. However, any modifications of the termini, along with what a skilled person envisages based on the state of the art in peptide chemistry, is also contemplated, as long as the resulting microprotein shows tryptase-inhibiting activity. In the closed conformation, the ends of the peptide backbone of the microproteins are connected, preferably via a covalent bond, more preferably via an amide (i.e. peptide) bond. Microproteins with a closed conformation having a cystine knot topology are known in the prior art as “cyclotides” and their knot as “cyclic cystine knot (CCK)”. Such cyclotides are for instance described in WO 01/27147 and Craik (Curr. Opinion in Drug Discovery & Development 5 (2002), 251-260).

It is furthermore preferred that the microproteins for use in the present invention comprise the amino acid motif CX₃-CX₄-CX₄₋₇-CX₁-CX₄₋₅-CX₅₋₇ (SEQ ID NO: 18), with X meaning independently from each other any amino acid residue. C means, in accordance with the standard nomenclature, cysteine. Preferably, the amino acids X are not cysteine. It is furthermore preferred that the cysteine residues C in that sequence form a cystine knot as defined above.

In accordance with a further preferred embodiment of the invention, the microprotein has a length of between 28 and 40 amino acids.

It has been shown in experiments conducted in connection with the present invention that microproteins not exceeding a certain maximum size show a particularly good performance, especially in regard to the capacity to bind all four catalytic sites of the tryptase tetramer. Accordingly, it is particularly preferred that the microproteins for use in connection with the present invention have a length of up to 35 amino acids, more preferably of up to 32 amino acids, and most preferably of up to 30 amino acids.

Furthermore, it is preferred that the microprotein for use in connection with the present invention and in accordance with the aforementioned definitions comprises an amino acid sequence selected from the group consisting of:

-   (a) the amino acid sequence depicted in any one of SEQ ID NOs: 1 to     15; -   (b) the amino acid sequence depicted in SEQ ID NO: 16 or 17; -   (c) a fragment of the amino acid sequence of (a) or (b), said     fragment being capable of inhibiting tryptase activity; and -   (d) a functional equivalent in which at least one residue of the     amino acid sequence or of the fragment of any one of (a) to (c) is     substituted, added and/or deleted, said functional equivalent being     capable of inhibiting tryptase activity.

The microproteins defined under (a) having the amino acid sequence of any one of SEQ ID NOs: 1 to 13 have been shown experimentally to efficiently inhibit tryptase (see Example 3 and Table 1, infra). Their use is therefore particularly preferred in connection with the present invention. The nucleotide sequence of a particularly preferred microprotein is shown in FIG. 5 (SEQ ID NO: 20). In addition, it is particularly preferred that the microprotein for use in connection with the present invention comprises the amino acid sequence of any one of SEQ ID NOs: 1, 14 and 15. These are the amino acid sequences of the microproteins McoTI-I, McoTI-II and McoTI-III described in Hernandez (2000).

The consensus sequence of SEQ ID NO: 16 referred to under (b) has been derived from the amino acid sequence of the microprotein MCoTI-KKV (SEQ ID NO: 7) which showed to have the highest tryptase inhibiting activity among the microproteins tested. It is conceived that at positions 1, 2, 8, 11, 12, 14 and 15 of SEQ ID NO: 16 amino acid residues lysine (K) or arginine (R) may reside interchangeably. The consensus sequence referred to under SEQ ID NO: 17 differs from that of SEQ ID NO: 16 in its C-terminal part.

The present invention also refers to the use of microproteins comprising a fragment of an amino acid sequence as defined in (a) or (b), provided said fragment has tryptase-inhibiting activity. The term “fragment” has a clear meaning to a person skilled in the art and refers to a partial continuous sequence of amino acid residues within the amino acid sequence with reference to which the fragment is defined. Thus, compared to the reference amino acid sequence, the fragment lacks at least one amino acid residue at the N-terminus, at the C-terminus or at both termini. In the case of a circular reference sequence, the fragment lacks at least one amino acid residue at one position of said sequence, whereby the fragment may be circular or linear. Preferably, the fragment retains the six conserved cysteine residues and, by their presence, is capable of forming the cystine knot topology.

The term “functional equivalent” refers to variants of a microprotein as defined in any one of (a) to (c), in which at least one residue of the amino acid sequence or the fragment of any one of (a) to (c) is substituted, added and/or deleted, said variant being capable of inhibiting tryptase activity. Preferably, the functional equivalent has an amino acid sequence which comprises six cysteine residues which are connected via disulfide bonds so as to form a cystine knot.

A functional fragment for use in the present invention may for example be a polypeptide which is encoded by a polynucleotide the complementary strand of which hybridises with a nucleotide sequence encoding a microprotein as defined in any one of (a) to (c), wherein said polypeptide has the activity of inhibiting tryptase activity.

In this context, the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA. In an especially preferred embodiment, the term “hybridization” means that hybridization occurs under the following conditions:

-   Hybridization buffer: 2×SSC; 10×Denhardt solution (Fikoll     400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na₂HPO₄;     -   250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA;     -   or     -   0.25 M of sodium phosphate buffer, pH 7.2;     -   1 mM EDTA     -   7% SDS -   Hybridization temperature T=60° C. -   Washing buffer: 2×SSC; 0.1% SDS -   Washing temperature T=60° C.

Polynucleotides encoding a functional equivalent which hybridize with a nucleotide sequence encoding a microprotein as defined in any one of (a) to (c) can, in principle, be derived from any organism expressing such a protein or can encode modified versions thereof. Such hybridizing polynucleotides can for instance be isolated from genomic libraries or cDNA libraries of bacteria, fungi, plants or animals.

Such hybridizing polynucleotides may be identified and isolated by using the polynucleotides encoding the microproteins described herein or parts or reverse complements thereof, for instance by hybridization according to standard methods (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA).

Such hybridizing polynucleotides also comprise fragments, derivatives and allelic variants of one of the polynucleotides encoding a microprotein as defined in any one of (a) to (c), as long as the polynucleotide encodes a polypeptide being capable of inhibiting tryptase. In this context, the term “derivative” means that the sequences of these polynucleotides differ from the sequence of one of the polynucleotides encoding a microprotein as defined supra in one or more positions and show a high degree of homology to these sequences, preferably within sequence ranges that are essential for protein function. Particularly preferred is that the derivative encodes an amino acid sequence comprising six cysteine residues which are connected via disulfide bonds so as to form a cystine knot.

The property of a polynucleotide to hybridize a nucleotide sequence may likewise mean that the polynucleotide encodes a polypeptide, which has a homology, that is to say a sequence identity, of at least 30%, preferably of at least 40%, more preferably of at least 50%, even more preferably of at least 60% and particularly preferred of at least 70%, especially preferred of at least 80% and even more preferred of at least 90% to the amino acid sequence of a microprotein as defined in any one of (a) to (c), supra. Moreover, the property of a polynucleotide to hybridize a nucleotide sequence may mean that the polynucleotides has a homology, that is to say a sequence identity, of at least 40%, preferably of at least 50%, more preferably of at least 60%, even more preferably of more than 65%, in particular of at least 70%, especially preferred of at least 80%, in particular of at least 90% and even more preferred of at least 95% when compared to a nucleotide sequence encoding a microprotein as defined in any one of (a) to (c), supra.

Preferably, the degree of homology is determined by comparing the respective sequence with the amino acid sequence of any one of SEQ ID NOs: 1 to 17. When the sequences which are compared do not have the same length, the degree of homology preferably refers to the percentage of amino acid residues or nucleotide residues in the shorter sequence which are identical to the respective residues in the longer sequence. The degree of homology can be determined conventionally using known computer programs such as the DNAstar program with the ClustalW analysis. This program can be obtained from DNASTAR, Inc., 1228 South Park Street, Madison, Wis. 53715 or from DNASTAR, Ltd., Abacus House, West Ealing, London W13 0AS UK (support@dnastar.com) and is accessible at the server of the EMBL outstation.

When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

Preferably, the degree of homology of, the hybridizing polynucleotide is calculated over the complete length of its coding sequence. It is furthermore preferred that such a hybridizing polynucleotide, and in particular the coding sequence comprised therein, has a length of at least 75 nucleotides and preferably at least 100 nucleotides.

Preferably, sequences hybridizing to a polynucleotide encoding a microprotein for use in connection with the invention comprise a region of homology of at least 90%, preferably of at least 93%, more preferably of at least 95%, still more preferably of at least 98% and particularly preferred of at least 99% identity to a polynucleotide encoding a specifically disclosed microprotein, wherein this region of homology has a length of at least 75 nucleotides and preferably of at least 100 nucleotides. Homology, moreover, means that there is a functional and/or structural equivalence between the compared polynucleotides or the polypeptides encoded thereby. Polynucleotides which are homologous to the above-described molecules and represent derivatives of these molecules are normally variations of these molecules having the same biological function. They may be either naturally occurring variations, preferably orthologs of a polynucleotide encoding a microprotein as defined in any one of (a) to (c), supra, for instance sequences from other alleles, varieties, species, etc., or may comprise mutations, wherein said mutations may have formed naturally or may have been produced by deliberate mutagenesis. The variants, for instance allelic variants, may be naturally occurring variants or variants produced by chemical synthesis or variants produced by recombinant DNA techniques or combinations thereof. Deviations from the polynucleotides encoding the above-described specific microproteins may have been produced, e.g., by deletion, substitution, insertion and/or recombination, e.g. by the fusion of portions of two or more different microproteins. Modification of nucleic acids, which can be effected to either DNA or RNA, can be carried out according to standard techniques known to the person skilled in the art (e.g. Sambrook and Russell, “Molecular Cloning, A Laboratory Manual”; CSH Press, Cold Spring Harbor, 2001 or Higgins and Hames (eds.) “Protein expression. A Practical Approach.” Practical Approach Series No. 202. Oxford University Press, 1999). Preferably, amplification of DNA is accomplished by using polymerase chain reaction (PCR) and the modification is used by appropriate choice of primer oligonucleotides, containing e.g. mutations in respect to the template sequence (see, e.g. Landt, Gene 96 (1990), 125-128). The polypeptides being variants of the concrete microproteins disclosed herein possess certain characteristics they have in common with said microproteins. These include for instance biological activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behavior in gel electrophoreses, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc.

The biological activity of the microproteins for use in connection with the invention, in particular the activity of inhibiting tryptase can be tested by methods as described in the prior art and in the Examples.

A suitable assay for tryptase inhibition activity is described in Example 3. The calculation of the apparent Ki-values (also designated Ki_(app)) which are indicative for the tryptase inhibiting activity of a given microprotein may be conducted according to Morrison (1969). This calculation is described in further detail for the analogous measurement of trypsin inhibition in Example 4. Typically, microproteins encompassed by the uses of the present invention have a tryptase inhibiting activity with a Ki of not more than 1 mM, preferably not more than 0.5 mM, more preferably not more than 0.2 mM, still more preferably not more than 0.1 mM, further preferred not more than 0.05 mM, particularly preferred not more than 0.02 mM, especially preferred not more than 0.005 mM. Most preferred is a Ki of not more than 0.002 mM. It is understood that the values determined in the activity assays may vary within an error range typical for the particular assay system applied, preferably within a range of +/−20%, further preferred with +/−10% and particularly preferred within 5%.

It is further preferred that a microprotein for use in connection with the present invention additionally shows an inhibitory activity on trypsin. As is outlined in Example 4 (infra), a test for trypsin inhibition may be indicative for the formation of the correct folding topology. A suitable trypsin inhibition assay is described in Example 4 (infra) which is based on the methods described in Van Nostrand (1990) and Sinha (1991). Preferably, the microproteins for use in connection with the present invention show a Ki for trypsin in the range of not more than 1 nM and preferably of not more than 0.5 nM. Advantageously, in view of a high selectivity for tryptase which may be desirable for therapeutic applications, it is preferred that the microproteins for the uses of the invention show a comparatively low inhibitory activity with regard to other proteases, such as trypsin or blood co-aggulation factors.

The term “tryptasen” includes the four closely related enzymes so far known which are α-, I-, II/β- and III-tryptase sharing a sequence identity between 90 and 98% (Miller, 1998; Vanderslice, 1990). Tryptase is the major secretory protease of human mast cells and is proposed to be involved in neuropeptide processing and tissue inflammation. Mature human tryptase is a tetrameric glycosylated molecule, is heparin-associated and composed of heterogenous, catalytically active subunits (see, e.g. Vanderslice et al., 1990; Miller et al., 1989, Sommerhoff et al., 1999). Tryptase is stored in mast cell secretory granules. After mast cell activation, human tryptase can be found in various biologic fluids. In connection with the present invention, the preferred target of the microproteins is mast cell tryptase, more preferably β-tryptase or α-tryptase.

The microproteins for use in connection with the present invention may consist solely of amino acids, preferably naturally occurring amino acids. However, encompassed are also microproteins which are derivatized in accordance with techniques familiar to one skilled in peptide and polypeptide chemistry. Such derivatives may for instance include the replacement of one or more amino acids with analogues such as chemically modified amino acids, the cyclisation at the N- and C-termini or conjugation with functional moieties that may for instance improve the therapeutical effect of the microproteins. The inclusion of derivatized moieties may, e.g., improve the stability, solubility, the biological half life or absorption of the polypeptide. The moieties may also reduce or eliminate any undesirable side effects of the microprotein. An overview for suitable moieties can be found, e.g., in Remington's Pharmaceutical Sciences by E. W. Martin (18^(th) ed., Mack Publishing Co., Easton, Pa. (1990)). Polyethylene glycol (PEG) is an example for such a chemical moiety which may be used for the preparation of therapeutic proteins. The attachment of PEG to proteins has been shown to protect them against proteolysis (Sada et al., J. Fermentation Bioengineering 71 (1991), 137-139). Various methods are available for the attachment of certain PEG moieties to proteins (for review see: Abuchowski et al., in “Enzymes as Drugs”; Hoicerberg and Roberts, eds. (1981), 367-383). Generally, PEG molecules are connected to the protein via a reactive group found on the protein. Amino groups, e.g. on lysines or the amino terminus of the protein are convenient for this attachment among others. Further chemical modifications which may be used for preparing therapeutically useful microproteins include the addition of cross-linking reagents such as glutaraldehyde, the addition of alcohols such as glycol or ethanol or the addition of sulhydroxide-blocking or modifying reagents such as phosphorylation, acetylation, oxidation, glucosylation, ribosylation of side chain residues, binding of heavy metal atoms and/or up to 10 N-terminal or C-terminal additional amino acid residues. Preferably, the latter residues are histidines or more preferably the residues RGS-(His)₆.

A further suitable derivatisation may be the fusion with one or more additional amino acid sequences. In such fusion proteins, the additional amino acid sequence may be linked to the microprotein sequence by covalent or non-covalent bonds, preferably peptide bonds. The linkage can be based on genetic fusion according to methods known in the art or can, for instance, be performed by chemical cross-linking as described in, e.g., WO 94/04686. The additional amino acid sequence may preferably be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker may comprise plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of the tertiary structure formed by the additional sequence and the N-terminal end of the microprotein or vice versa. The fusion protein may comprise a cleavable linker or cleavage site for proteinases (e.g., CNBr cleavage or thrombin cleavage site; see Example 4, supra).

Furthermore, said additional amino acid sequence typically has a predefined specificity or function, e.g., nuclear localization signals, transactivating domains, DNA-binding domains, hormone-binding domains, protein tags (GST, GFP, h-myc peptide, FLAG, HA peptide).

In a preferred embodiment, the microprotein is fused to barnase, preferably to inactive barnase.

“Barnase” is an extracellular ribonuclease from Bacillus amyloliquefaciens (Fersht, 1993; Paddon, 1987). It has been shown in connection with the present invention that the fusion of a microprotein to barnase can bring about a number of advantages. In particular, when the microprotein is produced recombinantly by the expression in a host cell, such as E. coli, the fused barnase moiety has solubilizing effect. This may greatly reduce or completely avoid the need to isolate the expressed microprotein from inclusion bodies and to subsequently oxidize it to obtain the active disulphide-bonded conformation. Further advantages lie in the possibility to use barstar-barnase affinity for purifying the expressed microprotein from the crude extract (see Example 5, infra) as well as in the feasibility to crystallize the fusion protein and to analyze the three-dimensional structure by using the known barnase structure as an input for a facilitated structure modeling (see Example 6).

If the barnase fusion is constructed using an active barnase, it may be necessary to co-express the barnase inhibitor barstar in sufficient amount since otherwise the barnase has a lethal effect on the host cell (Martsev, 2004). In view of this, it may be preferable to use an inactive mutant of barnase such as the one having His-102 replaced by Ala (see Example 4, infra). Thereby, the advantages connected with barnase fusions are maintained, while it is not necessary to additionally co-express barstar.

The microprotein for use in connection with the present invention may, e.g., be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). For the provision of the microprotein via recombinant expression, an overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440). An overview of yeast expression systems is for instance given by Hensing et al. (Antonie van Leuwenhoek 67 (1995), 261-279), Bussineau et al. (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991), 742-745) and Buckholz (Bio/Technology 9 (1991), 1067-1072).

Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence.

It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodríguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. Natl. Aced. Sci. USA (1983), 21-25), Ip1, rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of proteins. These promoters often lead to higher protein yields than do constitutive promoters. In order to obtain an optimum amount of protein, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

Transformation or transfection of suitable host cells can be carried out according to one of the methods mentioned above. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc. The microprotein can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

Depending upon the host employed in a recombinant production procedure, the expressed polypeptide may be glycosylated or may be non-glycosylated. The polypeptide may also include an initial methionine amino acid residue.

Preferably, the microprotein is first recombinantly produced as a fusion protein, advantageously with barnase, and then released from the fusion partner by cleavage at the fusion linkage and subsequent separation.

Likewise, the microprotein may be produced by any suitable standard peptide synthesis procedure as described in the art (see, e.g., Merrifield, Methods Enzymol. 289 (1997), 3-13; Hancock, Mol. Biotechnol. 4 (1995), 73-86; and Merrifield, Adv. Enzymol. Relat. Areas Mol. Biol. 32 (1969), 221-296), such as for instance that used in Example 1 (infra).

For administration to a subject, the microprotein may be formulated as a pharmaceutical composition. Such pharmaceutical compositions comprise a therapeutically effective amount of the microprotein and, optionally, a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered with a physiologically acceptable carrier to a patient, as described herein. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin (see supra). Such compositions will contain a therapeutically effective amount of the aforementioned microprotein, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In another preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The pharmaceutical composition for use in connection with the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

In vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Preferably, the pharmaceutical composition is administered directly or in combination with an adjuvant.

In the context of the present invention the term “subject” means an individual in need of inhibiting the activity of tryptase. Preferably, the subject is a vertebrate, even more preferred a mammal, particularly preferred a human.

The term “administered” means administration of a therapeutically effective dose of the aforementioned pharmaceutical composition comprising the microprotein to an individual. By “therapeutically effective amount” is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

The methods are applicable to both human therapy and veterinary applications. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %. The agents may be administered alone or in combination with other treatments. The administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intra-arterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, intrabronchial, transdermally, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the pharmaceutically effective agent may be directly applied as a solution dry spray.

The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

The dosages are preferably given once a week, however, during progression of the treatment the dosages can be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., daily. In a preferred case the immune response is monitored using methods known to those skilled in the art and dosages are optimized, e.g., in time, amount and/or composition. Progress can be monitored by periodic assessment. The pharmaceutical composition may be administered locally or systemically. Administration will preferably be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

In a preferred embodiment, the pharmaceutical composition is formulated as an aerosol for inhalation.

In a further preferred embodiment, the pharmaceutical composition is formulated for the oral route of administration.

In a preferred embodiment, the present invention refers to the-above-described use, wherein the microprotein is administered to the patient in the form of a gene delivery vector which expresses the microprotein. Furthermore preferred is that the cells are transformed with the vector ex vivo and the transformed cells are administered to the patient.

According to these embodiments, the pharmaceutical composition for use in connection with the present invention is a vector comprising and capable of expressing a polynucleotide encoding a microprotein as described above. Such a vector can be an expression vector and/or a gene delivery vector. Expression vectors are in this context meant for use in ex vivo gene therapy techniques, i.e. suitable host cells are transfected outside the body and then administered to the subject. Gene delivery vectors are referred to herein as vectors suited for in vivo gene therapeutic applications, i.e. the vector is directly administered to the subject, either systemically or locally. The vector referred to herein may only consist of nucleic acid or may be complexed with additional compounds that enhance, for instance, transfer into the target cell, targeting, stability and/or bioavailability, e.g. in the circulatory system. Examples of such additional compounds are lipidic substances, polycations, membrane-disruptive peptides or other compounds, antibodies or fragments thereof or receptor-binding molecules specifically recognizing the target cell, etc. Expression or gene delivery vectors may preferably be derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses or bovine papilloma virus, and may be used for delivery into a targeted cell population, e.g. into cells of the respiratory tract. Methods which are well known to those skilled in the art can be used to construct recombinant expression or gene delivery vectors; see, for example, the techniques described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2001) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the vectors can be reconstituted into liposomes for delivery to target cells. The vectors containing the a microprotein-encoding polynucleotide can be transferred into a host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see Sambrook, supra).

Suitable vectors and methods for ex-vivo or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The vectors for use in this embodiment of the invention may be designed for direct introduction or for introduction via liposomes or viral vectors (e.g. adenoviral, retroviral) into the cell. Preferred gene delivery vectors include baclovirus-, adenovirus- and vaccinia virus-based vectors. These are preferrably non-replication competent.

The use of the present invention preferably refers to a disease selected from the group consisting of asthma, inflammation, psoriasis, pulmonary fibrosis, an interstitial lung disease, rheumatoid arthritis, gingivitis, peridontitis, an allergic reaction, allergic rhinitis, osteoarthritis, atherosclerosis, angiogenesis, multiple sclerosis and cancer.

Due to their capacity to inhibit tryptase, the microproteins described herein-above can be utilized according to the present invention in order to prevent or treat diseases or conditions in which tryptase is a pathology-mediating agent. This refers in particular to mast cell-mediated inflammatory disorders. One aspect in this context especially refers to inflammatory diseases associated with the respiratory tract, such as asthma, psoriasis or allergic rhinitis. It is in particular contemplated to use microproteins for preventing or treating the late phase bronchoconstriction and airway hyperresponsiveness associated with chronic asthma. In addition, the use of the present invention refers to the treating of other types of immunomediated inflammatory disorders, such as psoriasis, rheumatoid arthritis, conjunctivitis as well as inflammatory bowel disease. A further preferred use refers to the use of microproteins against acute myeloid leukemia (AML) where it has been shown that the myeloblasts of these patients express alpha-tryptase in excess over beta-tryptase and secrete pro-alpha-tryptase constitutively (Sperr, 2001, 2002). The present invention also includes the use of the above-described microproteins as anti-inflammatory agents. In this function, the microprotein may be a component of creams for topical administration, e.g., to insect, snake or scorpion bites, or to skin affected by dermatitis.

In a further aspect, the present invention relates to a method for the treatment of an individual in need of inhibiting the activity of tryptase comprising administering to said individual an effective amount of a pharmaceutical composition comprising the microprotein as defined above or a polynucleotide encoding said microprotein and, optionally, a pharmaceutically acceptable carrier.

With regard to this embodiment, the above explanations, in particular concerning the formulation of pharmaceutical compositions, mode of administration and diseases, likewise apply.

In accordance with the aforesaid, the present invention also refers to the use of the microprotein as defined above or a polynucleotide encoding said microprotein for inhibiting tryptase activity. This embodiment may refer to tryptase inhibition in vivo or in vitro, preferably in vitro.

Another embodiment of the present invention relates to the use of the microprotein as defined above for purifying tryptase.

For this purpose, the microprotein is preferably bound to a solid support. The term “purifying” includes in this context also removing, isolating or extracting tryptase. The support may comprise any suitable inert material and includes gels, magnetic and other beads, microspheres, binding columns and resins. For carrying out the present embodiment, standard protocols for affinity purification of proteins known to a skilled person are applicable.

In a further aspect, the present invention relates to the use of the microprotein as defined above as a carrier molecule for tryptase or a derivative thereof.

This application may in particular refer to the use of the microprotein as a carrier molecule for tryptase and tryptase-related compounds, such as in creams, oils, powders or pills, to provide slow release of the bound components.

Also, the present invention relates to the use of microproteins as defined above for detecting and/or quantifying tryptase in a sample.

The quantification of tryptase levels, preferably human mast cell tryptase levels may, for example, be applicable for blood, nasal lavage fluids, tissues or food products. In connection with this application, the microproteins may be employed together with means of detection (for example radiolabel, antibodies, enzymes such as alkaline phosphatases, peroxidases and luciferases) that allow the accurate quantification of tryptase in the sample to be tested. Accordingly, the present invention refers to corresponding kits comprising one or more microproteins and, preferably, suitable detection means. Such kits may resemble radioimmunoassay or ELISA kits, with the proteins of the invention acting as binding molecules, instead of antibodies directed against tryptase. The detection of tryptase may in particular be used for the detection of mast cells.

Any technique common to the art may be used in a detection method according to the present embodiment and may comprise immunocytochemical and histological techniques, in which the microprotein may be used in combination with antisera (such as anti-McoTI-II antisera), or in which the molecule is directly coupled to a label or dye, such as a fluorescent dye, e.g. FITC. In another embodiment, the microprotein may be fused either genetically or synthetically to another protein such as an alkaline phosphatase, luciferase or peroxidase in order to facilitate its detection. Other methods to detect tryptase-containing cells or samples may involve blotting techniques (Towbin et al, 1979), gel retardation, affinity chromatography, or any of the other suitable methods that are used in the art.

Moreover, the present invention relates to a method for diagnosing a disorder associated with an aberrant abundance of tryptase in a given cell, tissue, organ or organism, comprising

-   (a) contacting a sample from said cell, tissue, organ or organism     with a microprotein as defined above under conditions allowing     binding between tryptase and the microprotein; -   (b) determining the amount of the microprotein bound to tryptase;     and -   (c) diagnosing a disorder when the determined amount is above or     below a standard amount.

In this context, the microprotein may be used in the form of a diagnostic composition which optionally comprises suitable means for detection. The microproteins described above can be utilized in liquid phase or bound to a solid phase carrier. Corresponding affinity assays may be carried out either in a competitive or a non-competitive fashion. Such affinity assays may be devised in a way analogous to the radioimmunoassay (RIA), the sandwich (immunometric assay) or the Western blot assay. The microproteins can be bound to many different carriers or used to isolate cells specifically bound to said polypeptides. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble.

There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.

The term “aberrant abundance” refers to a concentration of tryptase in a given cell, tissue, organ or organism which is significantly below or above a standard concentration of tryptase for said cell, tissue, organ or organism of a healthy individual so that it is associated with a disease to be diagnosed, preferably one of the diseases mentioned above. Preferably, the tryptase concentration when aberrantly abundant is reduced to not more than 75%, preferably not more than 50%, more preferably not more than 25%, and particularly preferred to not more than 10% of the standard concentration. Alternatively, the tryptase concentration in the aberrant state is preferably increased to at least 150%, more preferably to at least 200% and still further preferred to at least 500% of the standard concentration.

According to the above, the present invention also refers to the use of the microproteins as defined above or a polynucleotide encoding said microprotein for diagnosing a disease related to an aberrant expression of tryptase.

In a further aspect, the present invention also refers to a kit comprising a microprotein as defined above and a manual for carrying out the above-defined diagnostic method or the corresponding use and, optionally, means of detection or a standard tryptase sample.

The components of the kit of the present invention may be packaged in containers such as vials, optionally in buffers and/or solutions. If appropriate, one or more of said components may be packaged in one and the same container. Additionally or alternatively, one or more of said components may be adsorbed to a solid support such as, e.g., a nitrocellulose filter or nylon membrane, or to the well of a microtitre-plate.

A further embodiment of the present invention relates to a fusion protein comprising an inactive barnase.

The advantages of using a fusion protein comprising an inactive barnase have already been mentioned above in connection with the production of microprotein fusions. Accordingly, the fusion of a given protein to be expressed to inactive barnase can be summarized as follows:

-   (i) Fusion with barnase may lead to an improved solubility of the     protein to be expressed. This may be explained by a chaperone-like     effect the barnase has on its fusion partner. It is of, note that     this effect is observed irrespective of whether the barnase is fused     to the N- or to the C-terminus of the fusion partner. The     solubilizing function of barnase may facilitate the recombinant     production of a desired protein, in particular when it is to be     expressed in the cytoplasm of a host organism, wherein the host     organism preferably is a microorganism, advantageously a bacterium,     such as E. coli. For instance, the problem of resolving inclusion     bodies concomitant with a subsequent renaturation of the expressed     protein may be overcome by using a barnase fusion. The useful effect     of improving solubility of expressed proteins is already described     for active barnase (Martsev, 2004). -   (ii) A particular improvement associated with the use of inactive     barnase compared to an active one lies in the fact that the     experimental requirements for the expression of the fusion protein     are significantly reduced because it is no longer necessary to     co-express the barnase inhibitor barstar. Without its co-expression,     barnase activity has a lethal effect on the host cell (Martsev,     2004). Inactive barnase is already known in the art, e.g. from     Jucovic (1995). However, it could not have been foreseen whether an     inactivated barnase would show the advantages of the active barnase     in a fusion protein expression. This, however, has been shown     convincingly in the experiments described in Examples 2 and 4,     infra. The barnase fusion clones mentioned in Martsev (2004) to bear     functionally significant mutations in the barnase module are     preferably no subject-matter of the present invention. -   (iii) A further surprising advantage of a fusion with inactive     barnase is the fact that the expression product of this fusion can     easily be recovered from the crude preparation by applying the     strong binding interaction between barnase and barstar. Such an     approach has not yet been described for barnase fusions in general.     In addition, it would have been uncertain whether inactivated     barnase would indeed work in an affinity chromatography with barstar     as binding moiety. However, this has convincingly been proven in the     experiment described in Example 5, infra. -   (iv) Furthermore, based on the barnase moiety within the fusion     protein, the fusion protein can be combined non-covalently, but     nevertheless stably under physiological conditions with a second     fusion protein which comprises barstar. Thereby, bi- or multivalent     functions, such as for instance multiple microproteins, can be     combined within one structure. This principle is described in Deyev     (2003).

In a further aspect, the present invention also relates to fusion proteins comprising barnase and a microprotein. Preferably, said barnase is inactive.

Microproteins are known to a person skilled in the art. Preferred microproteins are in this context those which have been defined above in connection with the tryptase inhibiting function of microproteins.

With regard to the microprotein-barnase fusions, the same applies in regard to advantages as that outlined above under (i) to (iv). In addition, these fusion proteins also have the advantage of generally facilitating the recombinant expression of microproteins which, without the fusion to barnase, might not be expressible at all or only to an unsatisfactory extent. Furthermore, the barnase fusion may also facilitate the elucidation of the three-dimensional structure of a crystal from the microprotein. This aspect is described in more detail further below.

With regard to the construction principles and ways of production of the fusion proteins of the invention, it is herewith referred to corresponding standard techniques known to a person skilled in the art and, in particular, to the above explanations concerning fusion proteins set out in connection with the aspect of the invention concerning the therapeutical use of microproteins for inhibiting tryptase. It is preferred that the fusion proteins of the invention contain a cleavable linker between the barnase and the other portion so that the two portions may be readily separated from one another after expression. Corresponding linker sequences are well-known to the skilled person and examples thereof are mentioned herein. Furthermore, it is contemplated that the fusion protein of the invention may contain additional amino acid sequences along with the particular function the user intends.

A preferred embodiment of the present invention relates to nucleic acid molecules comprising a nucleotide sequence encoding the above-defined fusion protein of the invention.

The nucleic acid molecules of the invention can be any type of polynucleotide, e.g. DNA molecules or RNA molecules or combinations thereof. These polynucleotides can be obtained by any suitable technique known in the art, they, for instance, may be produced synthetically or by recombinant techniques, in vivo or in vitro, such as PCR. Such polynucleotides may comprise any modification thereof that is known in the state of the art (see, e.g., U.S. Pat. No. 5,525,711, U.S. Pat. No. 4,711,955, U.S. Pat. No. 5,792,608 or EP 302175 for examples of modifications). Such polynucleotides may be single- or double-stranded, linear or circular, without any size limitation. Preferably, the nucleic acid molecules are DNA, cDNA or mRNA.

The nucleic acid molecule encoding a fusion protein of the invention will generally be a recombinant nucleic molecule. The term “recombinant nucleic acid molecule” refers to any nucleic acid molecule that has been produced by a technique useful for artificially combining nucleic acid molecules or parts thereof that were beforehand not connected as in the resulting recombinant nucleic acid molecule. Suitable techniques are for example available from the prior art, as represented by Sambrook and Russell, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989).

In a preferred embodiment, the nucleic acid molecule comprised in the recombinant nucleic acid molecule is operably linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Suitable expression control sequences include promoters that are applicable in the target host organism. Such promoters are well known to the person skilled in the art for diverse hosts from the kingdoms of prokaryotic and eukaryotic organisms and are described in literature. For example, such promoters can be isolated from naturally occurring genes or can be synthetic or chimeric promoters. Likewise, the promoter can already be present in the target genome and may be linked to the coding sequence by a suitable technique known in the art, such as for example homologous recombination.

The present invention also relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering, that comprise a nucleic acid molecule of the invention.

In a preferred embodiment of the invention, the vectors of the invention are suitable for the transformation of fungal cells, plant cells, cells of microorganisms or animal cells, in particular mammalian cells. Preferably, such vectors are suitable for the transformation of microorganisms, such as yeast or bacteria, in particular of E. coli. Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook and Russell, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, the vectors may be liposomes into which the recombinant nucleic acid molecules of the invention can be reconstituted for delivery to target cells.

Advantageously, the nucleic acid molecules contained in the vectors and encoding a fusion protein of the invention are operably linked to one or more expression of the fusion protein in a host cell.

The expression of the nucleic acid molecules of the invention in prokaryotic or eukaryotic cells, for instance in Escherichia coli, may be interesting because it permits a more precise characterization of the biological activities of the proteins encoded by these molecules. In addition, it is possible to insert different additional mutations into the nucleic acid molecules by methods usual in molecular biology (see for instance Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), leading to the synthesis of proteins possibly having modified biological properties. In this regard, it is on one hand possible to produce deletion mutants in which nucleic acid molecules are produced by progressive deletions from the 5′ or 3′ end of the coding DNA sequence, and said nucleic acid molecules lead to the synthesis of correspondingly shortened proteins. On the other hand, the introduction of point mutations is also conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity of the protein.

For genetic engineering in prokaryotic cells, the nucleic acid molecules of the invention or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell, 2001, Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, “primer repair”, restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.

In a further embodiment, the invention relates to a method for producing cells capable of expressing a fusion protein according to the invention comprising genetically engineering cells with an above-described nucleic acid molecule, recombinant nucleic acid molecule or vector of the invention. Encompassed by the present invention are likewise cells obtainable by this method.

Another embodiment of the invention relates to host cells, in particular prokaryotic or eukaryotic cells, genetically engineered with an above-described nucleic acid molecule or vector of the invention, and to cells descended from such transformed cells and containing said nucleic acid molecule or vector of the invention and to cells obtainable by the above-mentioned method.

In a preferred embodiment the host cell is genetically engineered in such a way that it contains a nucleic acid molecule stably integrated into the genome. More preferably the nucleic acid molecule can be expressed so as to lead to the production of the encoded fusion protein.

An overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440). An overview of yeast expression systems is for instance given by Hensing et al. (Antoine von Leuwenhoek 67 (1995), 261-279), Bussineau (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antoine van Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991), 742-745) and Buckholz (Bio/Technology 9 (1991), 1067-1072).

Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication origin ensuring replication in the host selected, but also a bacterial or viral promoter and, in most cases, a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding nucleotide sequence. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), Ip1, rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of proteins. These promoters often lead to higher protein yields than do constitutive promoters. In order to obtain an optimum amount of protein, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose of IPTG (isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

The transformation of the host cell with a nucleic acid molecule or vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell, (Molecular Cloning: A Laboratory Manual (2001), Cold Spring Harbor Press, New York; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990). The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc. The protein according to the present invention can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromography and lectin chromatography. Depending on whether the protein is expressed intra- or extracellularly, the protein can be recovered from the cultured cells and/or from the supernatant of the medium. Protein refolding steps can be used, as necessary, in completing configuration of the protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

Accordingly, a further embodiment of the invention relates to a method for preparing a fusion protein comprising culturing the above-described host cells under conditions that the fusion protein encoded by the nucleic acid molecule with which said host cell is genetically engineered is expressed; and recovering the fusion protein from the culture.

In a particular preferred embodiment, the method of the invention for preparing a fusion protein comprises a step in which the fusion protein is purified by way of binding the barnase moiety of the fusion protein to barstar.

This application is in line with the above explanations according to which it is an advantageous property of the fusion protein of the invention that it can be purified by making use of the strong, but reversible binding activity between barnase and barstar. The feasibility of this principle has been demonstrated in Example 5, infra.

Moreover, the present invention relates to a fusion protein obtainable by the method for its production as described above.

The fusion protein of the present invention may be glycosylated or may be non-glycosylated.

The fusion protein of the invention may also include an initial methionine amino acid residue. The fusion protein according to the invention may be further modified to contain additional chemical moieties not normally part of the protein. Those derivatized moieties may, e.g., improve the stability, solubility, the biological half life or absorption of the protein. The moieties may also reduce or eliminate any undesirable side effects of the protein and the like. An overview for these moieties can be found, e.g., in Remington's Pharmaceutical Sciences (18^(th) edition, Mack Publishing Co., Easton, Pa. (1990)). Particular examples of suitable protein modifications are described above.

The present invention relates in a further embodiment to a method of detecting or purifying the fusion protein of the invention comprising the step of contacting the fusion protein with barstar. Preferably, in this method, barstar is immobilized. For immobilization any conceivable solid support may be used along with the particular intended application, and may include any suitable inert material such as gels, magnetic or other beads, microspheres, binding columns and resins. For detection purposes, barstar may preferably be coupled to a detectable moiety. Corresponding suitable moieties are described in the literature and examples are given above in connection with tryptase-detection and corresponding diagnostic methods.

Detection as well as purification may be carried out in accordance with techniques familiar to a skilled person.

Furthermore, the present invention also relates to the use of the fusion proteins of the invention for producing a crystal of said fusion protein.

In a further aspect, the present invention relates to a crystal of a microprotein fused with barnase.

The provision of the crystal of the invention is connected with a number of improvements over the prior art. For instance, the presence of the barnase in the microprotein fusion facilitates easy crystallizing of microproteins, apparently because barnase is good to crystallize. Heretofore, it was only in rare cases possible to achieve the crystallization of a solitary microprotein, i.e. without a bound target protein (e.g. trypsin) (Thaimattam, Acta Crystallogr. D. Biol. Crystallogr. 58 (2002), 1448-1461). The co-crystallization of a microprotein together with bound trypsin has been reported by Chakraborty (2001), Zhu (J. Protein Chem. 18 (1999), 505), Ay (Acta Crystallogr. D 59 (2003), 247) and Bode (FEBS L. 242 (1989), 285). Often crystallization of a microprotein is hampered because a target protein is not available in sufficient amounts or does not undergo crystallization. These difficulties have been overcome by the provision of the crystal of the invention since a solitary microprotein was shown to be well amenable to crystallization when it is fused to barnase (see Example 6, infra).

Moreover, it was surprisingly found that the presence of the barnase in the crystal does not influence the structure analysis of the fused microprotein.

A further advantage of the fusion to barnase lies in the fact that the already known barnase structure (Baudet, J. Mol. Biol. 219 (1991), 123-132; Martin, Acta Crystallogr. D. Biol. Crystallogr. 55 (1999), 386-398) may be utilized as a starting point for modelling the structure of the whole fusion protein, and thereby of the microprotein. The barnase structure data can be entered into the algorithm applied for determining the structure of the crystallized fusion protein. Thus, it is possible to resolve the structure of the microprotein without having any previous knowledge on its structure, because the diffraction data of the crystal can be interpreted starting from the known barnase structure.

The microprotein structure retrievable from the crystal of the invention may be of use for analyzing the topology of the binding site to the target molecule. Based on the information obtainable from such an analysis, it may for example be possible to optimize the binding of a microprotein by introducing chemical modifications in the structure of the microprotein, e.g. by changing one or more amino acid residues in the binding site. This has already been successfully practiced, for instance, with the microprotein EETI-II in connection with its inhibitory activity upon porcine pancreatic elastase (Hilpert, J. Biol. Chem. 278 (2003), 24986-24993).

Additionally, the elucidation of the microprotein structure promoted by the provision of the crystal of the invention may be beneficial for the construction of graftings onto microproteins. Microproteins are generally considered as being good carriers for peptide functions, such as peptides having a therapeutical effect. The introduction of such peptide sequences, also called “grafting”, into sites not essential for folding of the microprotein may for instance improve stability and bioavailability of the peptide in the subject to whom it is administered. Corresponding microprotein grafting approaches are for example described in WO 01/27147 and WO 01/34829. By the elucidation of the microprotein structure based on the crystal of the invention, the design of such grafting constructs may be improved. For example, the selection of suitable sites for introducing such a peptide into a microprotein may now be done more specifically. Also, microproteins comprising a grafted peptide may likewise be contained in a crystal of the invention and therefore their structure can be directly determined. This may, for example, yield a further optimization of the peptide display and target binding by rational design methods.

Such specific interventions are now greatly facilitated by the provision of the crystals of the invention and corresponding methods for producing them. A further advantage of the use of a fusion to barnase is the fact that this fusion protein can be readily purified by affinity chromatography using barstar as binding moiety.

The crystal of the invention may harbor any microprotein known to a person skilled in the art. Preferably, it is a microprotein defined above in connection with the uses of the microproteins for inhibiting tryptase. In a particularly preferred embodiment, the microprotein-barnase fusion contained in the crystal comprises the amino acid sequence set forth in SEQ ID NO: 19. It is furthermore preferred that the barnase contained in the crystal of the invention is inactive. In accordance with the explanations given further above, the use of an inactive barnase has the advantage that the co-expression of barstar can be omitted.

In a preferred embodiment, the crystal of the present invention belongs to space group C222₁. Furthermore, it is preferred that the crystal of the invention, has the unit cell dimensions of a=73.981 Å, b=217.820 Å and c=58.322 Å, α=β=γ=90°. A particularly preferred crystal of the invention has the crystal coordinates as depicted in Table 6, infra. Also preferred is a crystal the coordinates of which differ from the coordinates in Table 6 by a root mean square deviation of the C-alpha atoms by less than 3 Å, preferably by less than 2 Å and further preferred by less than 1 Å. Most preferably the crystal of the invention is obtainable by the method described in Example 6.

The term “crystal” as used herein refers to an ordered state of matter. Since it cannot be excluded that the crystallized proteins are not purified to homogeneity, corresponding impurities may be contained in the crystal. Even highly purified proteins may still be heterogeneous due to modifications, the binding of ligands or other effects. In addition, proteins are generally crystallized from complex solutions that may include not only the target molecule but also buffers, salts, precipitating agents and water. It is important to note that protein crystals are composed not only of protein, but also of a large percentage of solvents molecules, in particular water. The proportion of these other compounds within the crystal may vary from 30 to even 90%. Frequently, heterogeneous masses serve as nucleation centers and the crystals simply grow around them. The skilled person knows that some crystals diffract better than others. Crystals may vary in size from a barely observable 20 mm to 1 or more millimeters. Crystals useful for X-ray analysis are typically single, 0.05 mm or larger, and free of cracks and defects. However, advances in technology may allow analyzing increasingly smaller crystals.

The term “coordinate” as used herein, refers to the information of the three-dimensional organization of the atoms contained in the crystal of the invention which contribute to the protein structure. A common format for coordinate files is the so-called “PDP file format” (PDB=Protein Data Bank, http://www.pdb.org/) which is organized according to the instructions and guidelines given by the Research Collaboratory for Structural Bioinformatics (H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne: Nucleic Acids Research, 28 pp. 235-242 (2000)) and which allows a graphical representation by programs such as O (Jones et al. Acta Crystallogr. 1991, 47:110-111), rasmol (Trends Biochem Sci. 1995: 20(9):374), moiscript (Kraulis, P. (1991), J. Appl. Cryst. 24, 946-950), bobscript or Pymol (Delano, W. L. (2002), The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, Calif., USA).

The term “root mean square deviation” (RMSD) is used as a mean of comparing two closely related structures and relates to a deviation in the distance between related atoms of the two structures after structurally minimizing this distance in an alignment. Related proteins with closely related structures will be characterized by relatively low RMSD values whereas more changes will result in an increase of the RMSD value.

Another aspect of the present invention refers to a method of preparing the crystal of the invention comprising the steps of:

-   (a) providing an aqueous solution comprising a microprotein fused     with barnase; -   (b) providing a reservoir solution comprising a precipitating agent; -   (c) mixing a volume of said aqueous solution with a volume of said     reservoir solution thereby forming a mixed volume; and -   (d) crystallizing at least a portion of said mixed volume.

The growth of crystals may be effected according to standard protein crystallisation procedures described in the literature such as in DeLucas (J. Struct. Biol. 142 (2003), 188-206), Chemov (J. Struct. Biol. 142 (2003), 3-21), and McPherson (Structure 3 (1995), 759-768). In a preferred embodiment, the crystals are grown according to the method described in Example 6 (infra). Each of the steps identified in this protocol may be further and separately refined, as it will be apparent to the skilled practitioner.

The protein provided for crystallization should be sufficiently purified. Preparation and purification can be done according to conventional protocols such as those mentioned above in connection with expression techniques and vectors. Typically, a microorganism is transformed with an expression vector containing one or more copies of the gene encoding the respective microprotein-barnase fusion protein. The microorganisms, typically bacteria, may be grown under conditions that allow an optimized expression of the fusion protein. If the fusion protein is exported into the medium, it can be conveniently purified from the culture supernatant. Otherwise, the host cell has to be disrupted as the first step of the purification process. After recovery of a crude mixture from the cell culture, the fusion protein may be purified by using a combination of various purification steps. Some of these purification steps may be repeated, if appropriate. The fusion protein present in the crude extract is advantageously purified by employing a method comprising an affinity purification step. Such an affinity purification step may make use of the barnase-barstar binding interaction as already described above and in Example 5 (infra) in further detail. Alternatively, for example, antibodies raised against the microprotein or barnase may be used for the purification, wherein said antibodies (or fragments thereof) are preferentially coupled to a column material. Suitable antibodies may be obtained by conventional immunization protocols such as described, for example, in Harlowe and Lane, “Antibodies, A Laboratory Manual”, CHS Press, 1988, Cold Spring Harbor. The affinity purification step may be combined with gel filtration and/or anion exchange chromatography steps, before or after the affinity purification step. The purification protocol may further comprise one or more dialysis steps. The order of purification steps is preferably selected as described in Example 4 or 5.

The reservoir solution may be provided with suitable ingredients in accordance with standard crystallization techniques. The conditions for crystallization are provided by the reservoir solution which generally contains at least one compound selected from the group consisting of a buffer, a salt and a precipitant. The buffer is preferably MES, however, it may be replaced by any other buffer with a similar buffer capacity and pK_(I). The term “salt” refers to charged molecules composed of cation and anion and which are held together by ionic interactions. Preferably said salt contains molecules selected from the group consisting of Mg, Ca, Na, Cl, Br, I, Rb, P, S, K, Mn, Zn, Cu, B, Mo, Se, Si, Co, J, V, Ni, wherein these molecules are in their charged state and contain one or more counterions. The reservoir solution may additionally contain a detergent which is preferably selected from the group consisting of Triton X-100, NP 40 C₁₂E₉, Cl₂E₈, n-Dodecyl-β-D-maltoside, sucrose monolaurate, CTAB, Deoxy-BigChap, n-decyl-β-D-maltoside, Nony-β-D-glucoside, DDAO, n-Octanoylsucrose, MEGA-8, MEGA-9, IPTG, HEGA-8, HEGA-9, CHAPS, CHAPSO, BAM, CYMAL-2, C₁₄E₈, TWEEN and Brij59. The reservoir solution may further contain a reducing agent such as one selected from the group consisting of DTE, β-mercaptoethanol, cysteine and GSH.

Preferably, the reservoir solution comprises at least one compound selected from the group consisting of HEPES, NaCl, PEG 100, PEG 200, PEG 400, PEG 500, PEG 600, PEG 800, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 5000, PEG 6000, PEG 7000, PEG 8000, ammonium sulphate, ammonium acetate, sodium sulphate, organic solvents, isopropanol, citrate buffer, Tris buffer, cacodylate buffer, MES-buffer, dithiothreitol, octylglycopyranoside and uranylacetate.

As a preferred ingredience, the reservoir solution may contain PEG 400, advantageously at a concentration between 5 and 10%, more preferably at 7%. Further preferred ingrediences are ammonium sulphate, preferably at a concentration of 1 to 2 M, more preferably at 1.3 M, as well as MES buffer. Further preferred is that the reservoir solution has a pH between 6 and 7, preferably of 6.5. Crystallization is preferably carried out according to the sitting drop method, but other methods such as the hanging drop method are not excluded from the scope of the present invention. Generally, grown crystals may be cryoprotected prior to undergoing X-ray diffraction studies. Before freezing, e.g. in liquid nitrogen, the crystal is typically contacted with a cryprotectant such as a liquor containing 25% glycerol or a PEG and isopropanol-containing solution.

The person skilled in the art knows that additional factors such as temperature may be crucial for crystal formation. These and other conditions of crystallization as well as strategies to optimise conditions of crystallization are known by a skilled person and can be retrieved from the literature such as “Crystallization of Biological Macromolecules” by Alexander McPherson (Cold Spring Harbor Laboratory; 1st edition (1999).

The invention furthermore pertains to a crystal obtainable by the above-described method for preparing a crystal.

In a further embodiment, the invention relates to a method for determining the three-dimensional structure of a microprotein, comprising the steps of:

-   (a) performing an X-ray diffraction analysis of a crystal of a     microprotein fused with barnase; -   (b) computationally processing the diffraction data obtained in     step (a) in order to determine the three-dimensional structure of     the crystal components and thereby to obtain crystal coordinates;     and -   (c) storing the obtained crystal coordinates on a data storage     medium.

Preferably, step (b) further comprises to use data on the three-dimensional structure of barnase not being fused to the microprotein for modelling the structure of the barnase moiety within the fusion protein. Thereby it is possible to elucidate the microprotein's structure without any previous knowledge thereon. The barnase structure data is retrievable, e.g. from the RCSB PDB Web site (protein data bank, repository for the processing and distribution of 3-D biological macromolecular structure data) WWW.rcsb.org, entry 1A2P.

Preferentially, the crystal used in the method of the present embodiment is a crystal of the invention as defined above.

X-ray diffraction may be performed on a beamline such as the DESY beamline BW6. Data may be further processed with XDS (W. Kabsch, J. Appl. Cryst. 21, 67 (1988)) and scaled with XCALE (Kabsch, 1993). Further refinement is possible by applying CNS (A. T. Brünger et al. Acta Cryst D 54, 905 (1998)). The structure can finally be solved with, for example, AmoRe (J. Navaza, Acta Crystallogr. A 50, 157 (1994)) and analysed with Xfit (D. E. McRee, J. Struct. Biol. 125, 156 (1999)) while structure validatation may be performed with PROCHECK (R. A. Laskowski, M. W. MacArthur, J. Appl. Crystallogr. 26, 283 (1993)) and WHATCHECK (R. W. W. Hooft, G. Vriend, C. Sander, E. E. Abola, Nature 381, 272 (1996)). The final map containing the atomic coordinates of the constituents of the crystal may be stored on a data carrier, typically the data is stored in PDB format or in x-plor format, both of which are known to the person skilled in the art. However, crystal coordinates may as well be stored in simple tables or text files.

In a preferred embodiment of the present invention, the method for structure determination comprises additional steps of computer modelling, wherein computer modelling may include the step of (a) using virtual-screening tools for the search of compounds that bind to the microprotein; (b) using homology-modelling tools that search for compounds similar to known microprotein ligands and that make molecular contacts to the binding sites of the microprotein under investigation; (c) using molecular-modelling algorithms that allow an estimation of the binding affinities of compounds to the microprotein; or (d) using ligand construction tools that build up organic molecules that fit into the ligand binding site of the target molecule of the microprotein, such as tryptase or other proteases.

The design of molecules with particular structural relationships to part of a protein molecule are well established and described in the literature (see for example Cochran, A. G. (2000), Chem. Biol. 7, 85-94; Grzybowski, B. A., Ishchenko, A. V., Shimada, J., Shakhnovich, E. I. (2002), Acc. Chem. Res. 35, 261-269; Velasquez-Campoy, A., Kiso, Y., Freire, E. (2001), Arch. Biochem. Biophys. 380, 169-175; D'Aquino, J. A., Freire, E., Amzel, L. M. (2000), Proteins: Struc. Func. Genet. Suppl. 4, 93-107.). Any of these so-called “molecular modelling” methods for rational drug design can be used to find a ligand to a microprotein or a ligand to the microprotein target molecule that behaves analogously or similar to the microprotein. Most of these molecular modelling methods take into consideration the shape, charge distribution and the distribution of hydrophobic groups, ionic groups and hydrogen bonds in the site of interest of the protein molecule. Using this information, that can be derived from the crystal structure of proteins and protein-ligand complexes, these methods either suggest improvements to existing proposed molecules, construct new molecules on their own that are expected to have good binding affinity, screen through virtual compound libraries for such molecules, or otherwise support the interactive design of new drug compounds in silico. Programs such as GOLD (G. Jones, et al., Development and J. Mol. Biol., 267, 727-748 (1997)); FLEXX (B. Kramer et al., Structure, Functions, and Genetics, Vol. 37, pp. 228-241, 1999); FLEXE (M. Rarey et al., JMB, 261, 470-489 (1996)) DOCK (Kuntz, I. D. Science 257: 1078-1082, 1992); AUTODOCK (Morris et al., (1998), J. Computational Chemistry, 19: 1639-1662) are virtual screening programs designed to calculate the binding position and conformation as well as the corresponding binding energy of an organic compound to a protein. These programs are specially trimmed to allow a great number of “dockings”, that is calculations of the conformation with the highest binding energy of a compound to a binding site, per time unit. In this context, binding energy is not always a real value, but can be statistically related to a real binding energy through a validation procedure. These methods lead to molecules, termed here “hits” that have to be assessed by experimental biochemical, structural-biological, molecular-biological or physiological methods for their expected biological activity. Positively assayed molecules may thus constitute potential lead candidates for the design of bio-active compounds.

The terms “molecular modeling” and “molecular modeling techniques” refer to techniques that generate one or more 3D models of a ligand binding site or other structural feature of a macromolecule. Molecular modelling techniques can be performed manually, with the aid of a computer, or with a combination of these. Molecular modelling techniques can be applied to the atomic coordinates of a crystal of the present invention to derive a range of 3D models and to investigate the structure of ligand binding sites. A variety of molecular modelling methods are available to the skilled person for use in this regard (see e.g., G. Klebe and H. Gohlke, Angew. Chem. Int. Ed. 2002, 41, 2644-2676; Jun Zeng: Combinatorial Chemistry & High Throughput Screening, 2000, 3, 355-362 355; Andrea G Cochran, Current Opinion in Chemical Biology 2001, 5:654-659). At the simplest level, visual inspection of a computer model of the structure of a microprotein-barnase fusion can be used, in association with manual docking of models of functional groups into its binding pockets. Software for implementing molecular modelling techniques may also be used. Typical suites of software include CERIUS2 (available from Molecular Simulations Inc (http://www.msi.com/)), SYBYL (available from Tripos Inc; http://www.tripos.com), AMBER (available from Oxford Molecular; http://www.oxmol.co.uk/), HYPERCHEM (available from Hypercube Inc; (http://www.hyper.com/), INSIGHT II (available from Molecular Simulations Inc; http://www.msi.com/), CATALYST (available from Molecular Simulations Inc; http://www.msi.com/), CHEMSITE (available from Pyramid Learning; http://www.chemsite.org/), QUANTA (available from Molecular Simulations Inc; http://www.msi.com/). These packages implement many different algorithms that may be used according to the invention (e.g. CHARMm molecular mechanics). Their uses in the methods of the invention include, but are not limited to: (a) interactive modelling of the structure with concurrent geometry optimization (e.g., QUANTA); (b) molecular dynamics simulation of microprotein structures (e.g. CHARMM, AMBER); (c) normal mode dynamics simulation of microprotein structures (e.g. CHARMM). Modelling may include one or more steps of energy minimization with standard molecular mechanics force fields, such as those used in CHARMM and AMBER. These molecular modelling techniques allow the construction of structural models that can be used for in silico drug design and modeling.

Moreover, elucidation of the structure of another crystal according to the present invention but being different from the specific MCoTi-II-barnase crystal described herein (Example 6) may be facilitated by employing the three-dimensional structure of said specific crystal as a search model to locate the structure of the further microprotein-barnase fusion in its own crystal unit cell by rotation and translation searches. In particular, if a given microprotein-barnase fusion having a structure similar to MCoTi-II-barnase has been crystallized and X-ray diffraction data have been produced, the molecular replacement method can be applied. In this method, the three-dimensional structure of MCoTi-II-barnase is used as a search model to locate the unknown structure of the microprotein-barnase fusion in its own crystal unit cell by rotation and translation searches. This may avoid the cumbersome search for heavy atom derivatives.

Generally, the method of “molecular replacement” is applicable if the crystal structure of one protein A is known and if crystals of a homologous protein B have been obtained. X-ray diffraction data of crystals of protein B are collected and the three-dimensional model of protein A is used to search for its orientation (“rotation”) and position (“translation”) in the crystal unit cell or protein B, utilizing the X-ray diffraction data of the latter. If the search has been successful as indicated by certain correlation coefficients, the model of protein A may be refined against the X-ray data of the homologous protein B until convergence is achieved as indicated by the crystallographic reliability (R)-factor. As a result, the three-dimensional structure of protein B can be obtained.

In a further aspect, the present invention relates to the use of the crystal of the invention as defined above or structure data obtainable therefrom for designing or identifying a compound as a drug. Preferably, said drug is capable of inhibiting a serine protease, preferentially a tryptase.

In accordance with the above explanations in connection with the method of the invention for identifying the three-dimensional structure of a microprotein, the structure data may be used to design or identify new drugs which have the same target as the microprotein, but shows pharmacological advantages such as an improved selectivity or degradation within the body. Thereby, the three-dimensional structure of the microprotein allows identifying points of contact with the target molecule. Based on these data, a drug having the desired characteristics may be designed or identified, by applying computational means, as referred to above. In some cases, it may be advantageous to develop new ligands de novo, i.e. not on the basis and as a modification of a pre-existing compound. The term “de novo compound design” refers to a process whereby the binding pocket of the target macromolecule are determined, and its surfaces are used as a platform or basis for the rational design of compounds that will interact with those surfaces. The molecular modeling steps used in the methods of the invention may use the atomic coordinates of a crystal of the present invention and models or structures derived therefrom, to determine binding surfaces. In particular, the present invention also refers to structures, i.e. PDB-files, of a microprotein. Any such structure will preferably reveal van der Waals contacts, electrostatic interactions, and/or hydrogen bonding opportunities. Said binding surfaces will typically be used by grid-based techniques (e.g. GRID, CERIUS.sup.2; Goodford (1985) J. Med. Chem. 28: 849-857) and/or multiple copy simultaneous search (MCSS) techniques to map favorable interaction positions for functional groups. This preferably reveals positions in the binding pocket sites of the microprotein for interactions such as, but not limited to, those with protons, hydroxyl groups, amine groups, hydrophobic groups (e.g. methyl, ethyl, benzyl) and/or divalent cations. Based on this information, scaffolds may be generated which mimic the structure of the binding sites of the microprotein. These may comprise functional groups which are chemical groups that interact with one or more sites on an interaction surface of a macromolecule. Once functional groups which can interact with specific sites at the target molecule of the microprotein have been identified, they can be linked so as to form a single compound using either bridging fragments with the correct size and geometry or frameworks which can support the functional groups at favorable orientations, thereby providing a desired drug or lead compound for this. Whilst linking of functional groups in this way can be done manually, perhaps with the help of software such as QUANTA or SYBYL, the following software may be used for assistance: HOOK, which links multiple functional groups with molecular templates taken from a database, and/or CAVEAT, which designs linking units to constrain acyclic molecules. Other computer-based approaches to de novo compound design that can be used with the atomic coordinates of the present invention include LUDI (Bohm (1992) J. Comp. Aided Molec. Design 6: 593-606, SPROUT (available from http://chem.leeds.ac.uk/ICAMS/SPROUT.html) and LEAPFROG (available from Tripos Inc; http://www.tripos.com). Suitable in silico libraries include the Available Chemical Directory (MDL Inc), the Derwent World Drug Index (WDI), BioByteMasterFile, the National Cancer Institute database (NCI), and the Maybridge catalog. Compounds in these in silico libraries can also be screened for their ability to mimic the binding sites of the microprotein by using their respective atomic coordinates in automated docking algorithms.

Furthermore, the microprotein structure date may also be used for screening a compound that binds to a microprotein. An automated docking algorithm is one which permits the prediction of interactions of a number of compounds with a molecule having a given atomic structure. Suitable docking algorithms include: DOCK (Kuntz et al. (1982) J. Mol. Biol. 161: 269-288), AUTODOCK (Goodsell et al. (1990) Proteins: Structure, Function and Genetics 8: 195-202), MOE-DOCK (available from Chemical Computing Group Inc; http://www.chemcomp.com/) or FLEXX (available from Tripos Inc; http://www.tripos.com). Docking algorithms can also be used to verify interactions with ligands designed de novo.

The present invention further relates to a machine-readable data storage medium comprising the structure data of the crystal of the invention and which, when read by an appropriate machine, can be used to display a three-dimensional representation of the microprotein contained in the crystal or a portion thereof. Preferably, such a data storage medium is obtainable by the above-described method for determining the three-dimensional structure of a microprotein. The storage medium in which the structure may be provided is preferably random-access memory (RAM), but may also be read-only memory (ROM, e.g. a CDROM or DVD), a diskette or a hard drive. The storage medium may be local to the computer, or may be remote (e.g. a networked storage medium, including the internet). The recorded data are preferably the atomic co-ordinates as shown in Table 6. Any suitable computer can be used according to the present invention for representing or further processing the data recorded on the medium.

It is another aspect of the present invention to provide a device comprising (a) the above-defined machine-readable data storage medium; and (b) a computer program for the display of the 3-dimensional microprotein model; and optionally (c) software for the evaluation of potential ligands or, processed forms of the microprotein such as peptidomimetics.

It is yet another aspect of the present invention to provide the use of the device of the present invention for modelling a ligand to or a drug analogous to the microprotein.

In accordance with the above explanations, the present invention also refers to a method of identifying a compound capable of binding to a microprotein comprising the steps of:

-   (a) performing a fitting reaction by computational means between a     microprotein and a candidate compound using the structure data of     the microprotein obtainable from the crystal of the invention and     structure data of said compound; and -   (b) determining whether the candidate compound is capable of binding     the microprotein based on the data obtained in the fitting reaction.

Suitable methods for identifying a compound capable of binding microprotein have already been outlined in connection with the use of the present invention for designing or identifying a drug.

In a preferred embodiment, the present method also encompasses developing a ligand which comprises the step of modifying an identified ligand to alter, add or eliminate a portion thereof suspected of interacting with a binding site of the microprotein, thereby increasing or decreasing the affinity of the ligand to the binding site.

Accordingly, the present invention additionally relates to the use of the crystal of the invention or structure data obtainable therefrom or the data storage medium of the invention for identifying a compound capable of binding to a microprotein.

In a further aspect, the present invention refers to a method for optimizing the binding activity of a microprotein to a target molecule comprising the steps of:

-   (a) determining by computational means one or more residues of the     microprotein which participate in the binding interaction with the     target molecule using structure data of the microprotein obtainable     from a crystal according to the invention and structure data of the     target molecule; and -   (b) modifying in the microprotein one or more of said determined     residues so as to optimize the binding activity of the microprotein     to the target molecule.

Particularly preferred is that the structure data of the microprotein and the target molecule are obtained from a crystallized co-complex of said microprotein and target molecule.

It is furthermore preferred that the structure data of the microprotein the binding activity of which is to be optimized is obtained by computationally superimposing its structure on the structure data of another microprotein's crystal, which is preferably a crystal according to the present invention.

For this embodiment of the present invention, suitable techniques of the rational design may be applied. These are generally known to a person skilled in the art. “Optimizing” may refer to an increase or a decrease of the binding affinity to a given target molecule, as can be expressed for example by Ki-values (see supra), preferably an increase is meant. Alternatively, or in addition, “optimizing” may for example refer to an increased or decreased, preferably increased, selectivity for the target molecule. For example, the selectivity for β-tryptase may be increased by enhancing the binding affinity for this enzyme and concomittantly decreasing the affinity to other target molecules to which the microprotein under investigation shows significant binding, such as trypsin or other tryptase isoforms. In accordance with the present embodiment, the binding affinity of the microprotein can be significantly increased, preferably by orders of magnitude, by a series of rational measures known to the person skilled in the art. These include the modification of the microprotein with chemical groups so as to reduce their degrees of freedom lost upon binding to the target molecule site or the introduction of more potent electrostatic or hydrophobic binding groups.

Particularly preferred is to carry out the present embodiment, as follows:

In cases where the three-dimensional structure of the target and the ligand microprotein is known or, most preferably, where the structure of the co-complex of both compounds is known, predictions can be made to optimize the binding of the microprotein. As an illustration, it is herewith reported that for the McoETi tryptase inhibitor, the structure of McoETi was superimposed onto the structure of the EETI-II microprotein in complex with trypsin as is retrievable from the protein data bank www.rcsb.org, entry 1H9H. Next, the tryptase structure was superimposed to trypsin, making use of the software Insight II (Accelrys). As a result, a model of McoETI binding to tryptase was obtained. As expected, it was found that the side chain of lysine residue 5 contacts the catalytic serine 195 OH. Interestingly, the active site of tryptase contains a number of acidic residues and some of the basic residues of the McoETI inhibitor loop together with the two adjacent arginines seem to contribute to binding. Hence, a number of variants have been made, where either additional lysines were introduced at the amino-terminus or internal lysine/arginine residues were systematically replaced by alanine. By performing rational design in this way, a substantial improvement of affinity was thus achieved (see Table 1, infra). In this way, the microproteins depicted in Table 1 (infra) under items 8, 18, 19 and 20 (i.e. SEQ ID Nos: 4, 11, 12 and 13) have been produced.

According to the above, the present invention also relates to the use of the crystal of the invention or structure data derivable therefrom for optimizing the three-dimensional structure of said microprotein with respect to its binding and/or inhibiting activity to a target molecule, said target molecule preferably being a tryptase.

These pharmaceutical compositions may be formulated in an analogous manner as it is described above for the microprotein. A skilled person is aware of suitable ways for formulating proteinaceous compounds provided in crystallized form. The use of protein crystals in medicine is for example described in Gappa (Tissue Eng. 7 (2001), 35-44) and Brader (Nat. Biotechnol. 20 (2002), 800-804).

Accordingly, the present invention also relates to the use of a crystal of the invention for the preparation of a pharmaceutical composition for treating or preventing a disease that can be treated or prevented by inhibiting the activity of tryptase. Preferably, the disease is selected from the group consisting of asthma, inflammation, psoriasis, pulmonary fibrosis, an interstitial lung disease, rheumatoid arthritis, gingivitis, peridontitis, an allergic reaction, allergic rhinitis, osteoarthritis, atherosclerosis, angiogenesis, multiple sclerosis or cancer.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database “Medline” may be utilized which is available on the Internet, for example under http://www.ncbi.nim.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nim.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.ch/biology/research_tools.html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.google.de. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

Furthermore, the term “and/or” when occurring herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

FIG. 1 depicts the amino acid sequences of the McoTI-II cyclotide (SEQ ID NO: 22) (A), of an open chain variant of McoTi-II called McoTi-o (SEQ ID NO: 1) (B) and of a hybrid consisting of the aminoterminal part of McoTi-o and the ICK peptide EETI-II called McoEeTi (SEQ ID NO: 23) (C).

FIG. 2 shows a HPLC profile of crude linear peptide McoTI-o. Peak 3: linear target peptide. As products from incomplete coupling were detected: peak 1: missing Pro; peak 2: missing TyrCys; peak 4: additional ^(t)Bu group.

FIG. 3 is a profile of an HPLC purification of McoTi-o after oxidation of cysteine residues. The elution time and the absorption of the effluent at 217 nm are given on the abscissa and the ordinate, respectively. Peak 1 indicates the folded peptide McoTI-o, peak 2 a mixture of not complete or proper folded intermediates.

FIG. 4 illustrates the barnase′-ICK peptide fusion construct design. (A) Schematic representation of the plasmid pBar100 harboring the barnase′ gene that leads to the expression of the enzymatically inactive H102A variant; f1, replication origin; cat, chloramphenicol resistance marker; tetR, tetracycline repressor encoding gene; colE1, colE1 replication origin; tac, tac promotor sequence; phoAs, alkaline phosphatase periplasmic signal sequence; ICK, ICK peptide encoding sequence. (B) DNA and protein sequence of the barnase′-ICK peptide fusion. The exchanged amino acid at position #102 of barnase (H102A) is indicated in bold letters. In the pBar100 series of expression vectors, a single methionine codon resides at the junction of barnase′ and ICK peptide coding sequence that can be used for chemical cleavage of the fusion protein with cyanogen bromide. The pBar100Throm vector encodes in addition a thrombin recognition site (LVPRGS).

FIG. 5 depicts the nucleotide and corresponding amino acid sequence of a synthetic rMcoEeTi hybrid gene (SEQ ID NOs: 20 and 21).

FIG. 6 represents a schematic outline of ICK peptide production and purification as described in Example 2. As a first step, the bacterial liquid culture of E. coli cells containing the respective pBar vector is acidified with glacial acidic acid which results in the release of the barnase′-ICK peptide fusion into the culture medium. The filtrated culture supernatant is subjected to a combined cation exchange/reverse-phase HPLC chromatography. To obtain pure fusion protein, a second cation exchange chromatography is performed after solubilization in 0.1 M Tris-HCl pH 7.8 containing 8 M urea and dialysis against 50 mM ammonium acetate. Cleavage of the fusion with CNBr or thrombin results in the release of the ICK peptide from the barnase′ carrier and the ICK peptide is isolated by RP-HPLC.

FIG. 7: displays an SDS-PAGE analysis of the purification of rMcoEeTi described in Example 2. M: Protein molecular weight marker (MBI Fermentas) with sizes indicated in kDa.; lane 1: culture supernatant (1.6 ml, precipitated with TCA); lane 2: culture supernatant after acidification (1.6 ml, precipitated with TCA); lane 3: purified barnase′-ThromMcoEeTI fusion protein; lane 4: fusion protein after overnight cleavage with thrombin.

FIG. 8: shows an HPLC analysis of the barnase′-McoEeTI (A) and the barnase′-EETI-II M7I (B) peptide fusion prior to cleavage (upper trace) and after cleavage with CNBr or thrombin, respectively (lower trace). The experimentally determined molecular mass of the respective ICK peptide is given together with the calculated molecular mass (indicated in parentheses).

FIG. 9 shows the result of an assay for the inhibition of human tryptase by McoTi-KKV. Tryptase (0.2 nM) was preincubated with McoTi-KVV (0-34 nM) at 37 degree C. for 60 min, and the reaction was initiated by the addition of substrate tos-Gly-Pro-Arg-AMC. The residual steady state velocities were measured over 10 min and are expressed in % of the activity measured in the absence of the inhibitor.

FIG. 10 shows the result of an assay for the inhibition of human tryptase by McoEeTI. Tryptase (0.2 nM) was preincubated with MCoEeTI (0-1950 nM) at 37 degree C. for 60 min, and the reaction was initiated by the addition of substrate tos-Gly-Pro-Arg-AMC. The residual steady state velocities were measured over 10 min and are expressed in % of the activity measured in the absence of the inhibitor.

FIG. 11 shows the amino acid sequence and three-dimensional structure of the ICK peptide EETI-II (SEQ ID NO: 24), a trypsin inhibitor from Ecballium elaterium (Chiche, 1989). The ribbon representation of the secondary structure elements of mature EETI-II was drawn with the program PYMOL (http://www.pymol.org). Disulfide bonds are shown as sticks. Cysteine linkage is also indicated in the amino acid sequence, where the inhibitor loop is marked in bold letters.

FIG. 12 is a depiction of an SDS-PAGE onto which samples were loaded taken during a purification of a barnase′ fusion protein using barstar in the affinity column (see Example 5). S: Sample prior to column application; FT: Flow-through of sample applied to the column; W1: Wash 1; W2: Wash 2; W3: Wash 3; E1: Elution fraction 1; E2: Elution fraction 2; R1: Regeneration solution 1; and R2: Regeneration solution 2.

FIG. 13 shows a crystal of the barnase-MCoTi-II fusion used for data collection (see Example 6). Scale bat is 1 mm.

FIG. 14 illustrates the effect of crystal annealing. In house diffraction data before (left) and after (right) annealing recorded from the same crystal in approximately the same orientation. After annealing, spots are less smeared out and visible to higher resolution.

FIG. 15 shows the packing of the barnase-MCoTi-II crystal. Barnase and MCoTi-II of the first molecule are shown in blue and orange, respectively. Barnase and MCoTi-II of the second molecule are shown in green and yellow, respectively. FIG. 15A shows the packing of barnase as obtained by the molecular replacement. The C-terminal residue of barnase is shown in spheres. FIG. 15B shows the large solvent channels in the barnase-MCoTi-II crystal.

FIG. 16 gives an overall view of the barnase-MCoTi-II monomer. Barnase is shown with β-strands in blue and α-helices in red. The linker sequence (SSSM) is coloured orange, MCoTi-II is shown in green.

FIG. 17 presents an overlay of the two monomers of barnase-MCoTi-II in the asymmetric unit shown in red and blue. The monomers were aligned on the barnase. The Figure illustrates that the difference between the two monomers is due to a rigid body movement of barnase (lower left) and MCoTi-II (upper right) relative to one another around residues of the linker.

FIG. 18 presents an overlay of MCoTi-II from the two crystallographically independent molecules A (green) and molecule B (blue). In both monomers, Lys 10 is coordinated by a PEG molecule (shown in ball-and-stick representation), but the positioning of the PEG is different on both molecules.

FIG. 19 shows MCoTi-II from the A monomer in cartoon representation. Side chains of cysteines and Lys 10 are shown in ball-and stick representation. A PEG molecule is located next to the side chain of Lys 10 with its oxygens in hydrogen bonding distance to the terminal amino group of Lys 10.

FIG. 20 presents an overlay of the MCoTi-II monomer with the first state of the MCoTi-II NMR ensembles 1HA9 (A), 1IB9 (B) and with the high resolution crystal structure of the squash-type trypsin inhibitor MCTI-I (C). MCoTi-II is shown in green in all panels.

The following Examples illustrate the invention:

EXPERIMENTAL SET-UP Molecular Biological Techniques

Unless stated otherwise in the Examples, all recombinant DNA techniques are performed according to protocols as described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols.

Example 1 Chemical Synthesis of Open Chain McoTI Materials and Methods

All the chemicals used were of the highest grade available. Solvents were of analytical grade and used as supplied. N_(α)-Fmoc protected amino acids were used with the following side-chain protecting groups: t-Bu (Asp, Tyr), Boc (Lys), Trt (Cys, Asn), Pbf (Arg). Pseudo-proline dipeptide Fmoc-Asp(O^(t)Bu)-Ser(ψ^(Me,Me))pro-OH was purchased from Calbiochem-Novabiochem GmbH. ESI mass spectra were measured with a TSQ 700 Finnegan spectrometer. High-resolution ESI mass spectra were recorded with a Bruker APEX-Q III 7T. HPLC were performed on a Pharmacia Äcta basic system using YMC J'sphere ODS H-80, RP C-18 columns for preparative runs (250×4.6 mm, 4 μm, 80 Å) and for the analytical samples (250×4.6 μm, 80 Å).

Synthesis of the Linear McoTI-o

The linear peptide McoTi-o (see FIG. 1B; SEQ ID NO: 1) was assembled using a combination of automated and manual Fmoc-assisted SPPS on PEG-type amide NovaSyn® TGR resin (Calbiochem-NovaBiochem GmbH) with a loading capacity of 0.23 mmol/g. The C-terminal glycine (5 equiv. respective to resin loading) was activated by HATU/DIEA (5 equiv. and 10 equiv., respectively) and attached without pre-activation onto the resin within six hours. The loading with the first amino acid was determined UV-spectroscopically by monitoring of Fmoc deprotection (Chan et al., 2000). Chain elongation was performed using the Applied Biosystems peptide synthesizer ABI 433. Starting from 0.435 g (0.1 mmol) of amide resin, FastMoc 0.1 MPP CondMon mode with conditional monitoring was used for all amino acid cycles including pseudo-proline dipeptide, except cysteine. After pre-swelling first in DCM, then in NMP, each amino acid cycle has been programmed to include basically 16 min Fmoc deprotection with conditional conductivity monitoring, 8 min activation with HBTU/HOBt/DIEA; and 30 min coupling. Depending on the Fmoc-monitoring, additional 20 min of Fmoc deprotection and 50 min coupling followed by acetic anhydride capping were added to the regular cycles automatically.

Cysteine was coupled manually in DCM/NMP using the standard DIC/HOBt protocol (Albericio et al., 2000). The peptide-resin was transferred from the peptide synthesizer reaction vessel into an SPPS reactor and treated by nitrogen bubbling in the presence of activated cysteine until a negative Kaiser test was obtained. Then the resin was thoroughly washed with NMP and DCM and transferred back to the peptide synthesizer reaction vessel.

Cleavage of the peptide from the resin was achieved using TFA/DTT/TIS/anisole (23:1:0.5:0.5 v:w:v:v, 5 mL per 200 mg peptide-resin). The resin was gently shaken for 2.5 h, filtered, washed with TFA (3×1 mL). The combined filtrates were concentrated in vacuo, and the peptide was precipitated with MTBE. The peptide was centrifuged, washed with MTBE, dried, dissolved in water containing 0.1% TFA, and lyophilized. The crude, reduced peptide (18 mg, 66.6% calculated using the program of the ABI 433 peptide synthesizer) was purified using preparative RP-HPLC. The gradients of 0.1% TFA-water (eluent A) and 90% acetonitrile-0.1% TFA (eluent B) were employed with a flow rate of 10 ml/min within 30 min. The linear peptide was characterized by ESI mass spectrometry as well as by high-resolution ESI-FT-ICR-MS.

Folding Procedure

Oxidation of the linear McoTI-o to the cystine knot was performed by dissolving the reduced lyophilized peptide in 50 μl 10 mM HCl per mg of peptide followed by addition of NH₄HCO₃ (200 mM, pH 9.1) to a final concentration of 1-1.5 mg/ml (Wentzel et al., 1999). The reaction mixture was incubated overnight in a PET container under vigorous shaking at room temperature. Purification of folded peptide was done by RP-HPLC using Phenomenex C₁₈ columns (analytical: 250×4.60 mm; preparative: 250×10.00 mm). Conditions were as follows: Eluent A: H₂O containing 0.1% TFA, eluent B: 50% acetonitrile, 50% 2-propanol containing 0.1% (v/v) TFA. A linear gradient of 10-37% B was performed with flow rates of 1 ml/min for analytical purposes and 3.5 ml/min for preparative runs, respectively. With monitoring at 217 nm the oxidized peptide-containing fraction was collected and lyophilized. Successful oxidation was confirmed by ESI mass spectrometry and by measurement of inhibitory activity against trypsin, which is strictly dependent on correct disulfide bond formation (Wentzel et al., 1999).

Example 2 Recombinant Production in E. coli of McoEeTi

Construction of Expression Vector pBar100-McoETI

Two halves of the coding sequence for Bacillus amyloliquefaciens RNAse (Barnase) were separately PCR amplified with Taq polymerase (Eppendorf) using Bacillus amyloliquefaciens DNA and the primer pairs Barnase-up (5′-CCGGCGATGGCCATGGATGCACAG GTTATCAACACGTTTG-3′; SEQ ID NO: 25) and Barmitte-lo (5′-GTTCGTCCGCTTTTGCCCGGAAGT TTGCCTTCCCTGTTTGAG-3′; SEQ ID NO: 26) and Barmitte-up (5′-CTTCCGGGCAAAAGCGGACGAAC-3′; SEQ ID NO: 27) and Barnase-lo (5′-GAATTCGGTCTGA TTTTTGTAAAGGTCTGATAATGG GCCGTTGTTTTGTA-3′; SEQ ID NO: 28), respectively. The two halves of the resulting barnase′ coding sequence were combined by PCR using the oligonucleotide pair Barnase-up and Barnase-lo. This barnase′ PCR product was digested with Nco I and EcoR I and ligated with similarly cleaved pET22b (Novagen). From this vector, the barnase gene was PCR amplified using the primers bar′-Nhe-up (5′ GCGCACTAGTGCTAGCGATCTCGATCCCGCGAA 3′; SEQ ID NO: 29) and bar′-Sma-lo (5′ CTGTCCCGGGCGAATTCGGTCTGATTTTTGTAA AGGTCTGAT-AGGCGTCCGTTGTTTTG 3′; SEQ ID NO: 30), cleaved with Nhe I and Sma I and ligated into similarly cleaved pASK21-CKSend (Christmann et al., 1999). The resulting barnase′-EETI-II expression cassette was excised with Aat II and Pst I and ligated into similarly cleaved pMT416 (Hartley, 1988). The resulting plasmid contains a phoA leader sequence and the barnase′ gene under tac promotor control (Hartley, 1988). This expression cassette was then transplanted by cleavage with Ecl 136 II and Xba I and ligation into similarly cleaved pASKInt200, a derivate of pASKInt100 (Wentzel et al., 2001) that lacks the tetR promotor, to give pASKBarInt200. This plasmid was finally cleaved with Sap I and Sma I and overlapping DNA ends were filled up using T4 DNA Polymerase (MBI Fermentas). Blunt end ligation of the vector fragment resulted in pBar100-EETI-II M7I. The nucleotide and corresponding amino acid sequence of rMcoEeTi is shown in FIG. 5 and SEQ ID NOs: 20 and 21.

For construction of plasmid pBar100-McoEeTI, the McoEeTI gene was assembled with synthetic oligonucleotides cotiup2 (5′ GCATGCGCTCTTC-TAACTGCATATGCGG GCCCAACGGTTACTGCGGTTCCGGATCC 3′; SEQ ID NO: 31), cotilink (5′ CGTCGACATTTTTTCAG GATTTTCGGGCAAACACCA-CCGTCGGATCCGGAACCGCAG 3′; SEQ ID NO: 32) and cotilo (5′GCATGCGCTCTTCTGCAAGCACCCGGGCAGTCGGAGTCACGTCGACA-TTTTTTCAGG 3′; SEQ ID NO: 33) by polymerase chain reaction, assembled in vector pMalp-2 (New England BioLabs) and transplanted by cleavage with NcoI and Bsp120 I to similarly cleaved cloning vector pASKBar100-EETI-II M7I (see also FIG. 4).

Expression of Fusion Genes

The steps of expressing, purifying and cleaning the fusion protein are schematically outlined in FIG. 6. E. coli strain 71-18 [F′ lacl^(q) (lacZ.M15) proA+B+.(lac-proAB) supE thi1] (source B. Müller-Hill) containing helper plasmid pRep4 (Qiagen) was transformed with pBar100-McoEeTI by electroporation and grown in 50 ml dYT liquid media containing 25 μg/ml chloramphenicol and 37.5 μg/ml kanamycin overnight at 37° C. This culture was then used to inoculate 5 l of TB medium containing 25 μg/ml chloramphenicol and 37.5 μg/ml kanamycin in a 5 l fermenter (Bioengineering). Fermentation was performed at 30° C. At an O.D.₆₀₀ of 3-5 IPTG was added to a final concentration of 1 mM. After overnight incubation, the bacterial liquid culture was cooled to 4° C. and 55 ml of glacial acetic acid per liter of cell culture was added under continuous stirring. Acidification of the culture results in additional release of barnase′ fusion protein into the medium (Hartley, 1988). Stirring was continued for another 15 min followed by centrifugation at 4000 rpm for 60 min at 4° C. Insoluble particles were removed by filtration.

Purification of Fusion Proteins

The filtered culture supernatant was diluted 5-fold with H₂O and applied to a Perseptive HS20 cation exchance column (1.0×10 cm, 7.6 ml bed volume) mounted to a Vision™ BioCad workstation (PerSeptive Biosystems) at 8 ml/min. Elution was performed with a gradient ranging from 0 to 0.5 M NaCl. Peak fractions ranging from 166 mM to 500 mM NaCl were directly applied to a Perseptive R2 reversed phase column. After washing with H₂O/0.1% (v/v) TFA, the fusion protein was eluted from the column using a gradient from 5 to 50% acetonitrile/0.1% (v/v) TFA. Fusion protein containing fractions were combined and lyophilized. The fusion protein peptides was dissolved in 0.1 M Tris-HCl pH 7.8 containing 8 M urea, dialyzed overnight against 50 mM ammonium acetate and further purified by Perseptive HS20 cation exchance chromatography. Elution was performed with a gradient ranging from 90 to 330 mM NaCl. Barnase-McoETi containing fractions were dialysed against 50 mM ammonium acetate and lyophilised.

Thrombin Cleavage of Fusion Proteins

Lyophilized proteins were solubilized in 20 mM Tris-HCl pH 8.45, 150 mM NaCl and 2.5 mM CaCl₂ and 1 unit of thrombin from human plasma (Sigma, T-7009) per mg of fusion protein was added. The reaction was incubated overnight at 37° C. and analysed by SDS polyacrylamide gel electrophoresis (FIG. 7) or reversed phase HPLC using a Phenomenex C₁₈ column (Synergi 4u Hydro-RP 80A; 250×4.60 mm). Buffer A: H₂O containing 0.1% (v/v) trifluoroacetic add, buffer B acetonitrile containing 0.1% (v/v) trifluoroacetic acid. A linear gradient ranging from 10-37% buffer B was run at flow rate of 1 ml/min. Chromatography was monitored at 217 nm (FIG. 8). ICK peptide containing fractions were collected and lyophilized. The expected molecular mass of the McoETi peptide (3168.75) was confirmed by ESI mass spectrometry.

Example 3 Determination of Trypsin and Tryptase Inhibitory Activity Titration of the Inhibitor

The concentration of inhibitory active inhibitor was determined by titration with trypsin. Therefore, bovine pancreatic trypsin was standardised by active-site titration using p-Nitrophenyl p′-guanidinobenzoate (Chase & Shaw, 1970). The concentration of active inhibitor was calculated assuming a 1:1 interaction between the inhibitor and trypsin.

Determination of Equilibrium Constants:

Apparent equilibrium dissociation constants (Ki_(app)) for the complexes of the inhibitor with trypsin and tryptase were determined essentially as described by Bieth (Bull. Eur. Physiopathol. Respir. 16 (Suppl.) (1980), 183-197). Briefly, increasing concentrations of an inhibitor were incubated with a constant concentration of an enzyme. Substrate was then added, and the residual enzyme activity measured. Ki_(app)-values were calculated by fitting the steady state velocities to the equation for tight binding inhibitors (Morrison, 1969) using non-linear regression analysis. FIGS. 9 and 10 show tryptase inhibition curves for the microproteins McoTi-KKV and McoEeTi taken according to the above-described method.

In the following Table 1, an overview is provided of microproteins that have been produced in accordance with the procedure described in Example 1 or 2. Note that the microproteins for which neither “Barnase-Fusion” nor “Chemical Synthesis” is indicated have been expressed as a barnase fusion and the microprotein afterwards cleaved off by CNBr and purified.

Microproteins showing an inhibitory activity upon tryptase of above a Ki_(app) of 1 mM can in principle not be considered as having a therapeutic utility. In some cases, the measurement of the inhibitory effect on tryptase apparently showed no activity at all. In other cases, there was inhibitory activity, however, only above a Ki_(app) of 1 mM.

Barnase- Chemical Kiapp [82 mol/l]- Kiapp [μmol/l]- No. Substance New Nomenclature Fusion Synthesis Sequence Trypsin β3-Tryptase 1 SE-MC-K SE-MC x GVCPKILKKCRRDSDCPG 0.0003 0.02 ACICRGNGYCG (SEQ ID NO: 1) 2 SE-MA-0 SE-MC-TR-010 x GVCPAILKKCRRDSDCPG no inhibition 110 ACICRGNGYCG (SEQ_ID NO:34) 3 SE-MG-0 SE-MG GVCPKILKKCRRDSDCLA 0.0003 0.1 GCVCGPNGFCGS (SEQ ID NO:2) 4 SE-BMG-0 SE-MG (Bar) x Barnase′- 0.0002 0.1 SSSMGVCPKILKKCRRDS DCLAGCVCGPNGFCGS (SEQ ID NO:3) 5 SE-BME x Barnase′- no inhibition no inhibition SSSMGIEGREERICPLIW MECKRDSDCLAGCVCGPN GFCGS (SEQ ID NO:35) 6 SE-EM-0 SE-ET GCPRILIRCKQDSDCLAG 0.0001 1.3 CVCGPNGFCGS (SEQ ID NO:36) 7 MCoEeTI-2R-10 SE-MG-TR-010 (Bar) x Barnase′- 0.03 no inhibition* SSSMGVCPRNRCKCRRDS DCLAGCVCGPNGCGS (SEQ ID NO:37) 8 MCoEeTI-3R-3 SE-MG-TR-020 (Bar) x Barnase′- 0.0002 0.03 SSSMGVCPRILRRCRRDS DCLAGCVCGPNGFCGS (SEQ ID NO:4) 9 MCoEeTI-QRT-7 SE-MG-TR-030 (Bar) x Barnase′- 0.001 no inhibition* SSSMGVCPRNRQRCRRDS DCLAGCVCTNNKFCGS (SEQ ID NO:38) 10 MCoEeTI-KKV-1 SE-MG-TR-040 (Bar) x Ba′- 0.0002 0.02 SSSMGKKVGVCPKILKKC RRDSDCLAGCVCGPNGFC GS (SEQ ID NO:5) 11 MCoEeTI-ARD-2 SE-MG-TR-050 (Bar) x Barnase′- 0.0002 no inhibition* SSSMGVCPKILKACARDS DCLAGCVCGPNGFCGS (SEQ ID NO:39) 12 MCoEeTI-LKA-7 SE-MG-TR-060 (Bar) x Bar′- 0.0002 no inhibition* SSSMGVCPKILKACRRDS DCLAGCVCGPNGFCGS (SEQ ID NO:40) 13 MCoEeTI-T-1 SE-MG-TR-080 (Bar) x Barnase′- 0.0001 0.15 SSSMGVCPKILKKCRRDS DCLAGCVCTNNKFCGS (SEQ ID NO:6) 14 MCoTI-KKV SE-MC-TR-020 x KKVGVCPKILKKCRRDSD 0.0004 0.002 CPGACICRGNGYCG (SEQ ID NO:7) 15 MCoTI-R SE-MC-TR-030 x GVCPRILKKCRRDSDCPG 0.0008 0.02 ACICRGNGYCG (SEQ tD NO:8) 16 MCoEeTI SE-MG-TR-090 GSVCPKILKKCRRDSDCL ≈0.00003 0.20 AGCVCGPNGFCGS (SEQ ID NO:9) 17 MCoTI-LAKC SE-MC-TR-040 x GVCPKILAKCRRDSDCPG 0.0002 0.1 ACICRGNGYCG (SEQ ID NO:10) 18 MCoTI-LKAC SE-MC-TR-050 x GVCPKILKACRRDSDCPG 0.0004 0.03 ACICRGNGYCG (SEQ ID NO:11) 19 MCoTI-LRKC SE-MC-TR-060 x GVCPKILRKCRRDSDCPG 0.0005 0.01 ACICRGNGYCG (SEQ ID NO: 12) 20 MCoTI-LKRC SE-MC-TR-070 x GVCPKILKRCRRDSDCPG 0.0004 0.01 ACICRGNGYCG (SEQ ID NO:13) 21 MCoTI-AlaG SE-MC-TR-080 x GVCP(AlaG)ILKKCRRD 1 ≈10 SDCPGACICRGNGYCG (SEQ ID NO:41) 22 MCoEeTI-ARDs SE-MG-TR-110 (Bar) x Barnase′- SSSMGVCPKILKKCARDS DCLAGCVCGPNGFCGS (SEQ ID NO:42) 23 MCoEeTI-RAD SE-MG-TR-120 (Bar) x Barnase′- SSSMGVCPKILKKCRADS DCLAGCVCGPNGFCGS (SEQ ID NO:43) 24 MCoEeTI-KKV- SE-MG-TR-130 (Bar) x Barnase′- 3R SSSMGKKVCPRILRRCRR DSDCLAGCVCGPNGFCGS (SEQ ID NO:44) Table 1 gives an overview of the microproteins produced and tested in connection with the present invention. *, at 0.5 μM protein concentration.

Three microproteins, among which the best inhibiting one (i.e. SE-MC-TR-020), were tested for tryptase selectivity. The results of these binding assays are shown in Table 2. As can be seen, SE-MC-TR-020 has a good selectivity for tryptase and does not inhibit other proteases apart from trypsin, or only to a significantly reduced degree when compared to tryptase inhibition.

TABLE 2 K_(iapp) [nM] Protease SE-MC-TR- SE-MC- Family Protease Species SE-MG 020 TR-030 Tryptase β-III Human 100 2 20 β-Ia Human 70 3 40 β-II Human 70 4 40 ST2 Sheep 4000 100 1000 Pankreas Trypsin Porcine 0.3 0.4 0.8 proteases Chymotrypsin Porcine >1000 >1000 >1000 Elastase Porcine >1000 >1000 >1000 Koagulation Thrombin Human >1000 >1000 >1000 Faktor XA Human >1000 >1000 >1000 Fibrinolysis Plasmin Human ~600 ~400 >1000 tPA Human >1000 >1000 >1000 Neutrophiles Elastase Human >1000 >1000 >1000 Cathepsin G Human >1000 >1000 >1000 Kallikreines pK1 Human >1000 >1000 >1000 hK3 Human >1000 >1000 ~100

Example 4 Recombinant Production of Microproteins Fused to Barnase, Using the Barnase Moiety as a Purification Handle Materials and Methods

Enzymes were obtained from NEB or MBI Fermentas. All chemicals used were of the highest grade available. Solvents were of analytical grade and used as supplied. ESI mass spectra were measured with a TSQ 700 Finnegan spectrometer. Liquid chromatography was performed on a Vision BioCad workstation (PerSeptive Biosystems). RP-HPLC was done on a Kontron or an Äcta (Pharmacia Biotech) basic HPLC system.

Construction of pBar100 Vectors

A schematic representation of expression vector pBar100 vector and additional sequence information of the construct is given in FIG. 4. Plasmids pBar100-EETI-II M7I, pBar100-EETI-II, pBar100-McoEeTI and pBar100ThromMcoEeTI were constructed as follows: The 5′ and the 3′ half of the barnase coding sequence were separately PCR amplified with Taq polymerase (Eppendorf) using Bacillus amyloliquefaciens DNA and the primer pairs barnase-up (5′-CCGGCGATGGCCATGGATGCACAG GTTATCAACACGTTTG-3′; SEQ ID NO: 45), barmitte-lo (5′-GTTCGTCCGCTTTTGCCCGGAAGT TTGCCTTCCCTGTTTGAG-3′; SEQ ID NO: 46) and barmitte-up (5′-CTTCCGGGCAAAAGCGGACGAAC-3′; SEQ ID NO: 47), barnase-lo (5′-GAATTCGGTCTGA TTTTTGTAAAGGTCTGATAATGG GCCGTGTTTTGTA-3′; SEQ ID NO: 48), respectively. Oligonucleotides were designed such that a Sma I site at codon #63 is eliminated and active site histidine codon #102 is converted to alanine. This mutation leads to an inactivation of the ribonuclease activity of the resulting barnase′ protein (Jucovic, 1995).

The two halfs of the barnase′ coding sequence were combined by PCR using the oligonucleotide pair barnase-up and barnase-lo. The resulting barnase′ PCR product was digested with Nco I and EcoR I and ligated with similarly cleaved pET22b (Novagen). From this vector, the barnase gene was PCR amplified using the primers bar′-Nhe-up (5′ GCGCACTAGTGCTAGCGATCTCGATCCCGCGAA 3′; SEQ ID NO: 49) and bar′-Sma-lo (5′ CTGTCCCGGGCGAATTCGGTCTGATTTTTGTAA AGGTCTGATAGGCGTCCGTTGTTTTG 3′; SEQ ID NO: 50), cleaved with Nhe I and Sma I and ligated into similarly cleaved pASK21-CK^(send) (Christmann, 1999). The resulting barnase-EETI-II expression cassette was excised with Aat II and Pst I and ligated into similarly cleaved pMT416 (Hartley, 1988) kindly provided by L. Leveneki (University of Marburg, Germany). The resulting plasmid contains a phoA leader sequence and the barnase′ gene under tac promotor control (Hartley, 1988). This expression cassette was then transplanted by cleavage with Ecl 136 II and Xba I and ligation into similarly cleaved pASKInt200, a derivate of pASKInt100 (Wentzel, 2001) that lacks the tetR promoter, to give pASKBarInt200. The resulting plasmid was finally cleaved with Sap I and Sma I and overlapping DNA ends were filled up using T4 DNA polymerase (MBI Fermentas). Blunt end ligation of the vector fragment resulted in pBar100-EETI-II M7I.

pBar100-EETI-II M7I was used as a template for the construction of pBar100-EETI-II. To this end, the EETI-II encoding gene was PCR amplified from pBar100-EETI-II M7I with primers Nco-XA-eti-up (5′ GACTCCGGCCATGGGGATCGAGGGAAGGGGGTGCCC GCGCATTCTGATGCGCTGCAAACAGGACTC 3′; SEQ ID NO: 51) and cat-hind-mittelo (CCACAAGCTTGAAAACGTTTCAG, SEQ ID NO: 52). The resulting product was cleaved with Nco I and Hind III and ligated into similarly digested pBar100-EETI-II M7I. For construction of plasmid pBar100-McoEeTI, the McoEeTI gene was assembled with synthetic oligonucleotides cotiup2 (5′ GCATGCGCTCTTCTAACTGCATATGCGGGCCCAACGGTTACTGCGGTTCCGGAT CC 3′; SEQ ID NO: 53), cotilink (5′ CGTCGACATTTTTTCAG GATTTTCGGGCAAACACCACCGTCGGATCCGGAACCGCAG 3′; SEQ ID NO: 54) and cotilo (5′GCATGCGCTCTTCTGCAAGCACCCGGGCAGTCGGAGTCA-CGTCGACATTTTTTCAGG 3′; SEQ ID NO: 55) by polymerase chain reaction, assembled in vector pMalp-2 (New England BioLabs) and transplanted by cleavage with NcoI and Bsp120 I to similarly cleaved cloning vector pASKBar100-EETI-II M7I.

Plasmid pBar100-ThromMcoEeTI was constructed as described above for pBar100-EETI-II using pBar100-McoEeTI as a template and primers NcoThromMCoTi-up (5′ AGCTCTTCCATGGGGCTGGTTCCGCGTGGGTCCGTTGCCCGAAAATCCTGAAA AAATG 3′; SEQ ID NO: 56), which encodes a thrombin protease recognition sequence, and cat-hind-mittelo (SEQ ID NO: 52).

Fusion Protein Production

E. coli strain 71-18 [F′ lacl^(q) lacZΔM15 proA⁺B⁺ Δlac-proAB supE thi1] (source B. Müller-Hill) containing helper plasmid pRep4 (Qiagen) which contains a lac1 gene was transformed with pBar100-EETI-II M7I, pBar100-EETI-II, pBar100-McoEeTI and pBar100-ThromMcoEeTI, respectively by electroporation and grown overnight at 37° C. in 50 ml rich media containing 25 μg/ml chloramphenicol and 37.5 μg/ml kanamycin. This culture was then used to inoculate 5 l of TB medium containing 25 μg/ml chloramphenicol and 37.5 μg/ml kanamycin in a 5 I fermenter (Bioengineering) or 1 l of TB medium in a 1 I fermenter (Biostat M, Braun Biotech). TB medium contains 12 g bacto-tryptone, 24 g bacto-yeast extract, 4 ml glycerol and 100 ml of a sterile solution of 0.17 M KH₂PO₄, 0.72 M K₂HPO₄ per liter. Fermentation was performed at 30° C. At an O.D.₆₀₀ of 3 to 5, IPTG was added to a final concentration of 1 mM. After overnight incubation, the bacterial liquid culture was cooled to 4° C. and 55 ml of glacial acetic acid per liter of cell culture was added under continuous stirring. Acidification of the culture results in release of barnase′ fusion protein into the medium (Hartley, 1988). Stirring was continued for another 15 min followed by centrifugation at 4000 rpm for 60 min at 4° C. Insoluble particles were removed by filtration.

Purification of Fusion Proteins

The filtered culture supernatant was diluted 5-fold with H₂O and applied to a Perseptive HS20 cation exchance chromatography column (1.0×10 cm, 7.6 ml bed volume) mounted to a Vision BioCad workstation (PerSeptive Biosystems) at a flow rate of 8 ml/min. Elution was performed with a gradient ranging from 0 to 0.5 M NaCl. Peak fractions from 166 mM to 500 mM NaCl were directly applied to a Perseptive R2 reverse-phase column. After washing with H₂O/0.1% (v/v) TFA, the fusion protein was eluted from the column using a gradient from 5% (v/v) to 50% acetonitrile/0.1% (v/v) TFA. Fusion protein containing fractions were combined and lyophilized. For the purification of barnase′-ThromMcoEeTI, the fusion protein was pre-adsorbed to phosphocellulose prior to cation exchange chromatography as described (Hartley, 1972). Briefly, phosphocellulose P11 (Whatman) was treated with 0.5 N NaOH, H₂O, 0.5 N H₂SO₄ and 0.1 M Na-citrate as described and added to the acidified culture supernant (0.25 g/l liquid culture). After stirring at room temperature for 30 min, the phosphocellulose was filled into a 15×4 cm column, washed with 0.01 M ammonium acetate and the bound proteins were eluted with 2 M ammonium acetate. The protein solution was further concentrated with a rotation evaporator, lyophilized and re-solubilized in 25 mM Na-acetate pH 5.0 containing 8 M urea and applied to the combined cation exchange/reverse-phase HPLC (see above). Finally, lyophilized fusion protein was re-solubilized in 0.1 M Tris-HCl pH 7.8 containing 8 M urea, dialyzed overnight against 50 mM ammonium acetate and re-applied to the HS20 cation exchance column. Elution was performed with a gradient ranging from 90 to 330 mM NaCl.

CNBr Cleavage of Fusion Proteins

Lyophilized proteins were solubilized in 20 μl 70% formic acid per milligram of protein. Per milligram of fusion protein 0.6 μl of 5 M cyanogen bromide solution (Fluka) was added. After overnight incubation in a Teflon vial with a screw cap, the sample was either lyophilized or diluted 1:10 with 5% acetonitrile containing 0.1% (v/v) H₂O/TFA and directly used for RP-HPLC.

Thrombin Cleavage of Fusion Proteins

Lyophilized proteins were solubilized in 20 mM Tris-HCl pH 8.45, 150 mM NaCl and 2.5 mM CaCl₂ and 1 unit of thrombin from human plasma (Sigma, T-7009) per milligram of fusion protein was added. The reaction was incubated overnight at 37° C. and analysed by SDS polyacrylamide gel electrophoresis or RP-HPLC.

Purification of ICK Peptides

For separation of cleaved ICK peptide variants from the barnase′ carrier protein standard RP-HPLC was performed on a Kontron or an Äcta (Pharmacia Biotech) basic HPLC system using a Phenomenex C₁₈ column (Synergi 4u Hydro-RP 80Å; 250×4.60 mm). Buffer A: H₂O containing 0.1% (v/v) trifluoroacetic acid, buffer B: acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Linear gradients of 10-37% buffer B were run with flow rates of 1 ml/min. Chromatography was monitored at 217 nm, ICK peptide containing fractions were collected and lyophilized.

Determination of K_(I) Values

The inhibitory activity of the protein variants was measured according to van Nostrand et al. (1990) and Sinha et al. (1991). The remaining activity of bovine trypsin (Serva) was determined after incubation with the respective inhibitor for 10 min at 37° C. by monitoring hydrolysis of the chromogenic substrate Boc-Leu-Gly-Arg-pNA (Bachem, solubilized in Me₂SO) at 405 nm. Assays were performed in a final volume of 100 μl in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl₂ at 37° C. containing 5 nM of trypsin, 1.5 nM substrate and varying amounts of inhibitor (10⁻¹⁰-10⁻⁶ M). For calculation of K_(lapp)-values, v_(max) values were determined and the curve fitting tool of SigmaPlot (Jandel Scientific) was applied on the basis of the following formula, that describes the case of competitive tight binding inhibitors (Morrison, 1969):

$\frac{v_{1}}{v_{0}} = {1 - \frac{\left\lbrack E_{t} \right\rbrack + \left\lbrack I_{t} \right\rbrack + K_{iapp} - \left\{ {\left( {\left\lbrack E_{t} \right\rbrack + \left\lbrack I_{t} \right\rbrack + K_{iapp}} \right)^{2} - {{4\left\lbrack E_{t} \right\rbrack} \cdot \left\lbrack I_{t} \right\rbrack}} \right\}^{\frac{1}{2}}}{2\left\lbrack E_{t} \right\rbrack}}$

v_(i)=v_(max) value of inhibited reaction v_(o)=v_(max) value of uninhibited reaction [E_(t)]=total enzyme concentration (free and in complex) [I_(t)]=total inhibitor concentration (free and in complex) K_(lapp)=apparent dissociation constant of the enzyme-inhibitor complex.

Results and Discussion Choice of Carrier Protein and Production Strategy

Barnase, an extracellular ribonuclease from Bacillus amyloliquefaciens is a well-characterized enzyme which has been extensively used in structure-function studies Fersht, 1993; Paddon, 1987). Barnase is a relatively small protein with a M_(r) of approximately 12300 that can be produced in Escherichia coli via secretion into the periplasmic space and the culture medium in yields up to 100 mg of bacterial liquid culture (Hartley, 1988). It is composed of only 110 amino acid residues, contains no cysteine residues that might negatively interfere with disulfide bond formation of the fused ICK peptide and in addition it also contains no CNBr cleavable methionine residue. Furthermore, barnase can easily be purified by cation exchange chromatography (Hartley, 1988). To abolish the intrinsic RNAse activity of barnase we have constructed an enzymatically inactive variant (Jucovic, 1995) where the active site residue histidine #102 is replaced by alanine to give barnase′. Expression vector pBar100 which is shown in FIG. 4A contains the barnase′ gene under tac promoter control and the respective ICK peptide coding sequence that was placed in frame to the barnase′ gene together with a short linker sequence (FIG. 4B).

Fusion Protein Production and Purification

Four different ICK peptide variants have been successfully expressed in E. coli via fusion to barnase′ that are derived from the Ecballium elaterium trypsin inhibitor EETI-II (Chiche, 1989). McoEeTI is a hybrid ICK peptide that is composed of the 13 aminoterminal residues of EETI-II and the 16 carboxyterminal residues of the Momordica cochinchinensis trypsin inhibitor McoTI-II (Hernandez, 2000). Sequences and yields of purified cystine knot protein variants are shown in Table 3.

TABLE 3 ICK peptides expressed as fusions to barnase′. Fusion protein Kiapp^(b) ICK peptide Peptide sequence yield^(a) [mg] [M] EETI-II M7I GCPRILIRCKQDSDCLAGCVCGPNGFCGS 12 n.d. (SEQ ID NO:57) EETI-II GCPPILMRCKQDSDCLAGCVCGPNGFCGS 6.7 9.0 +− 4.2 × 10-9 (SEQ ID NO:58) McoEeTI GVCPRILKKCRRDSDCLAGCVCGPNGFCGS 24.3 7.1 +− 0.9 × 10-8 (SEQ ID NO:23) ThromMcoEeTI GSVCPRILKKCRRDSDCLAGCVCGPNGFCGS 7.4 n.d. (SEQ ID NO:59) ^(a)yield corresponds to the amount of purified fusion protein obtained from 11 E. coli culture. ^(b)inhibition constants were determined by incubation of varying amounts of the respective ICK peptide with trypsin and monitoring the residual trypsin activity using the trypsin substrate Boc-Leu-Gly-Arg-pNA (Wentzel, 1999). n.d.: not determined.

The underlying strategy for expression and purification is outlined in FIG. 6. Barnase′-ICK fusion proteins are directed into the periplasm of the host cell, where folding of the fusion protein and intramolecular disulfide bond formation of the ICK peptide can occur in the oxidative extracellular milieu. Acidification of the culture with glacial acidic acid results in the complete release of the barnase′-fusions from the periplasmic space into the culture medium (Hartley, 1988) (FIG. 7). The diluted culture medium is applied to cation exchange chromatography as a first purification step. Alternatively, to reduce the sample volume, the fusion protein can be pre-adsorbed to phosphocellulose followed by batch elution with ammonium acetate and lyophilization (Hartley, 1972) (data not shown). Subsequent RP-HPLC results in the removal of small peptides derived from tryptone and yeast extract of the culture medium. On average, 12 mg fusion protein have been obtained from 1 liter of bacterial liquid culture (Table 3). No efforts have been made yet to enhance protein yield via optimization of fermentation conditions towards high cell densities or by placing the barnase′ gene under control of a promoter that is stronger than the tac promoter used in these studies.

It is important to check whether the cystine knot peptides obtained are fully oxidized and contain the desired three pairs of disulfide bonds, where the first cysteine residue in the sequence is connected to the fourth, the second to the fifth, and the third to the sixth (FIG. 11). The ICK peptides of this study are trypsin Inhibitors and inhibition of trypsin activity can be used as a sensitive probe of correct folding, since correct disulfide connectivity is strictly required for high affinity trypsin binding (Wetzel, 1999). As can be seen in Table 3, both EETI-II and McoEeTI act as inhibitors of bovine trypsin as barnase′ fusion proteins and display strong inhibitory activity.

Fusion Protein Cleavage and Isolation of the ICK Peptides

A unique methionine residue was introduced at the junction of barnase′ and the respective ICK peptide. As a consequence, the ICK peptide can be released from the fusion protein by cyanogen bromide cleavage. As can be seen in FIG. 8B, the released ICK peptide elutes from a C18 column at lower acetonitrile concentrations than the barnase′ carrier, from which it can easily be separated. After overnight incubation of the fusion protein with CNBr in 70% formic acid at least 80% of the fusion was completely cleaved as judged by gel electrophoresis (data not shown). The expected molecular mass of the resulting fully oxidized cystine knot protein was confirmed by ESI-MS (FIG. 8B).

CNBr cleavage cannot be applied for the removal of the barnase′ carrier if the particular ICK peptide to be processed contains internal methionine residues as it is the case for the EETI-II ICK peptide. To have an alternative to CNBr cleavage at a methionine residue, expression vector pBar100ThromMcoEeTI was constructed, which gives rise to the synthesis of a fusion protein with a recognition sequence for the site-specific protease thrombin at the junction of barnase′ and the ICK peptide McoEeTI. Thrombin cleavage was performed in 20 mM Tris-HCl buffer pH 8.45 containing 150 mM NaCl and 2.5 mM CaCl₂ at 37° C. and was almost complete after overnight incubation (FIG. 7) The ICK peptide was isolated by reverse-phase HPLC and the expected molecular mass of Throm-McoEeTI was confirmed by ESI-MS (FIG. 8A).

Conclusions and Outlook

Peptides of the inhibitor cystine knot family share a common architecture that is defined by a combination of three intramolecular disulfide bonds. These interesting molecules show diverse biological activities including for example neurotoxins (Narasimhan, 1994) and enzyme inhibitors (Hamato, 1995; Chakraborty, 2001). Because of their enormous intrinsic stability and the possibility to apply methods of molecular evolution for the isolation of variants with predefined binding capabilities (Wentzel, 1999; Baggio, 2002), cystine knot proteins are considered to be ideal scaffolds for drug design (Craik, 2001). We have devised an expression and purification strategy that is based on ICK peptide synthesis via fusion to barnase′. Expression of functional barnase is lethal to the host cell and requires co-expression of the barnase inhibitor, barstar (Hartley, 1988). Our approach to use an enzymatically inactive variant obviates this necessity and may in addition allow one to purify the barnase′ fusion in a simple single step procedure via affinity chromatography on immobilized barstar, provided that the barnase H102A active site variant retains sufficient affinity to bind the inhibitor (see also Example 5, infra).

Recently, barnase fusions of antibody V_(L) domains and single chain Fv fragments have been described (Martsev, 2004; Deyev, 2003). A solubilizing effect of barnase was observed both in vitro and in vivo, which was explained by a chaperone-like role that barnase may exert on the fused antibody domain (Martsev, 2004). It appears as if barnase′ fusion generally supports folding and correct disulfide bond formation of the fused ICK peptide since we have also successfully expressed a number of other cystine knot peptides having a sequence different to that of the EETI-II and McoEeTI peptides shown in this Example. For these, we observed similar expression levels and high yields of folded peptides forming a cystine knot (see Example 3, supra).

In conclusion, the fusion protein system described here using barnase′ as a carrier for secretion and export and as a convenient purification handle allows for the production of folded cystine knot proteins that can be directly used for biological assays. If required, the barnase′ moiety can be removed, for example by chemical cleavage with cyanogen bromide or enzymatic cleavage with thrombin and the disulfide-bond containing ICK peptide can be isolated without the requirement of in vitro oxidation which often becomes the yield limiting step of cystine knot peptide production (Martsev, 2004).

Example 5 Purification of Microprotein-Barnase Fusion Using Immobilized Barstar

Barstar was purified as described (Jones et al., 1993, FEBS Lett. 331, p. 165). The purified protein was covalently immobilized to a Affi-gel matrix (BioRad). To this end, 20 mg of Barstar in PBS buffer was coupled to 1 ml AffiGel (BioRad) as described by the manufacturer. To show that Barnase can be purified by affinity chromatography via binding to immobilized barstar, the column was equilibrated with 50 mM ammonium acetate pH 7. A sample solution (S) containing the fusion protein consisting of barnase′ (the inactive mutant of barnase; see Example 3, supra) and the cystine knot microprotein SE-MG in PBS-buffer was applied to the column. The flow through (FT) was collected for analysis. The column was washed three times with 2.5 ml 50 mM ammonium acetate (W1, W2, W3). Bound protein was eluted by applying two times 2.5 ml 50 mM ammonium acetate, 8 M urea to the column (E1, E2). By addition of urea, both barstar and barnase are denatured and the barnase elutes from the column. The column was regenerated with 5 ml 50 mM ammonium acetate pH 7, 4 M urea (R1) followed by 5 ml 50 mM ammonium acetate pH 7 (R2). Aliquots of each fraction were analysed by SDS-polyacrylamide gel electrophoresis (see FIG. 12). The column could be reused at least three times (not shown).

The results of this experiment prove that tight interaction between barnase and barstar can be applied in order to purify fusion proteins comprising barnase. It is of particular note that this experiment has been carried out using an inactive variant of barnase. This means that the corresponding fusion protein can advantageously be expressed without the co-expression of barstar as it is described in the prior art to be necessary to avoid death of the host cell (see, e.g., Martsev, 2004; Deyev, 2003).

Example 6 Crystallization of a Microprotein and Structure Analysis Protein Crystallization and Data Collection

Initial screening using Jena Bioscience screens yielded small crystals under several conditions. Condition C3 from screen 6 (2 M ammonium sulphate, 5% PEG 400, 01.M MES pH 6.5) was further optimised and crystals used for data collection were grown by the sitting drop method at 4° C. with 1.3 M ammonium sulphate, 7% PEG 400 (v/v), 0.1 M MES pH 6.5 as reservoir solution. Droplets were mixed from 8 ml protein (30 mg/ml) and 4 ml reservoir solution. Crystals were plate shaped and grew over several weeks to a final size of about 500×500×35 mm (FIG. 13). Typically, stacks of plates were obtained, that had to be broken apart to isolate single plates for data collection. For cryoprotection crystals were immersed in mother liquor containing 25% glycerol and flash frozen in liquid nitrogen. Before data collection, crystals were annealed three times by blocking the cryo stream for several seconds. Annealing improved spot shape and resolution (FIG. 14). Data were collected to a resolution of 1.3 Å at DESY beamline BW6 at 100 K and a wavelength of 1.050 Å using a MarCCD detector. Data were processed with XDS and scaled with XSCALE (Kabsch 1993). Data statistics are given in Table 4. The raw data are given in PDB format in Table 6.

Structure Solution and Refinement

Initial phases were determined using in-house data to 2.9 Å by molecular replacement as implemented in the program EPMR (Kissinger, Gehlhaar et al. 1999) using a monomer of barnase (PDB ID 1A2P) as search model. Matthews probability predicted the asymmetric unit to most likely contain three molecules based on all entries in the PDB or four molecules given the resolution of the crystal (Kantardjieff and Rupp 2003). The search for three molecules in the asymmetric unit in EPMR yielded the highest correlation coefficient and lowest R factor upon placing the second molecule, while adding a third molecule gave worse results. Packing was checked in 0 (Jones, Zou et al. 1991). Barnase monomers packed against each other with their C-terminus pointing towards empty space available to the MCoTi-II part of the fusion protein (FIG. 15A). This indicated a correct solution and means that the crystals actually contain only 2 molecules per asymmetric unit. Accordingly, the Matthew's coefficient is 3.76 and the solvent content 67.3% (FIG. 15B), which is very unusual for crystals diffracting to 1.3 Å. Rigid body refinement of barnase was carried out in Refmac (Winn, Murshudov et al. 2003) followed by restrained refinement. An attempt to place the MCoTi-II part by molecular replacement using EPMR with barnase fixed and one state of the MCoTi-II NMR structure (PDB ID 1HA9) as search model failed. Hence, the MCoTi-II part was built manually using the initial 2.9 Å data. The model refined at 2.9 Å was used later for refinement with the high resolution data set keeping the free R set. ARP/wARP was used for finding water molecules. The current model contains all residues of the fusion protein, 477 water molecules, 2 PEG molecules, 6 sulfate ions and 3 glycerol molecules. Each barnase and each MCoTi-II was defined as a separate group for TLS refinement in REFMAC. Statistics of refinement are given in Table 4. Structure analysis, fitting of homologous structures and calculation of rmsds was carried out in Swiss PDB Viewer (Guex and Peitsch 1997). Figures were rendered in PyMol (DeLano 2002).

Results

A variant of the squash trypsin inhibitor MCoTi-II, which naturally occurs as a cyclic peptide in Momordica cochinchinensis (spiny bitter cucumber), was crystallized as a fusion protein with a catalytically inactive mutant of barnase, barnase′ (see Example 4, supra). The fusion protein (SEQ ID NO: 17) had been recombinantly expressed in E. coli and purified to homogeneity (Example 4, supra). The structure of the fusion protein (FIG. 16) was solved at a resolution of 1.3 Å by molecular replacement using the barnase wild-type structure as search model. There are two molecules of the fusion protein in the asymmetric unit. The two molecules can be aligned with an rmsd of 0.71 Å for 572 backbone atoms and an rmsd of 0.85 for all atoms (FIG. 17). The difference is mainly caused by a rigid body movement of barnase and MCoTi-II relative to one another, as the individual domains in the fusion protein can be overlaid with considerably lower rmsd (FIG. 18) 0.3 Å for 120 backbone atoms in MCoTi-II and 0.34 Å for 436 backbone atoms in barnase (see Table 5). The two proteins are joined by a four residue linker (SSSM) which allows for this flexibility. In both molecules of the asymmetric unit, a PEG molecule is found near Lys 10 of MCoTi-II (FIGS. 18 and 19). By analogy with the crystal structure of the complex formed between bovine beta-trypsin and MCTI-A, another squash-type trypsin inhibitor, Lys10 is the residue, which is inserted into the active site of trypsin. Although in different overall orientation, the PEGs in both molecules adopt a conformation similar to crown ethers, positioning four to six oxygens favourably for hydrogen bond formation. Lys 10 is among the residues varying most between the two MCoTi-II monomers. The largest differences are in residues Gly6 to Leu12 and around Pro29. When overlaid with the first states of two NMR structures available in the PDB (1HA9 and 1IB9) and with the crystal structure of MCTI-I (1F2S), another squashtype trypsin inhibitor, the agreement is best with the crystal structure of MCTI-I (FIG. 20).

Table 4 provides an overview of the data statistics for data collection and refinement statistics carried out when analyzing the crystal structure of fusion protein McoTi-II-barnase′

Data collection Space group C222₁ Cell dimensions a = 73.981, b = 217.820, c = 58.322 α = β = γ = 90°. Resolution range (Å)  40-1.3 (1.4-1.3) Measured reflections 356629 (59959)  Unique reflections 114401 (2246)  Completeness (%) 98.7 (98.1) R_(ivm)  3.9 (41.8) I/σI 14.01 (2.42)  Refinement Atoms Protein 2292 Water 477 PEG, sulfate, glycerol 94 R_(cryst)/R_(free) (%)¹⁾ 18.7/20.8 R.m.s. deviation from ideal Bonds (Å) 0.008 Angles (°) 1.569 Most favored/additionally allowed Φ, ψ (%) 91/9  ¹⁾The test set comprised 2292 reflections (2%).

Table 5 is a comparison of the two crystallographically independent fusion proteins contained in the asymmetrical unit of the McoTi-II-barnase′ fusion

# of atoms (bb = backbone Molecules compared Rmsd (Å) a = all atoms) Fusion protein 0.71 572 0.85 1102 Barnase 0.34 436 0.51 867 MCoTi-II 0.3 120 0.79 209

Table 6 (next pages) depicts the raw data obtained by X-ray diffraction structure analysis of the McoTi-II-barnase′ crystal described herein. The data is in PDB format. The two monomers contained in the asymmetrical unit are annoted in the data set as “A” and “B”. The coordinates of barnase′ including linker have order numbers 1-114, those of the microprotein portion 115-144.

HEADER  ---- XX-XXX-XX   xxxx COMPND  --- REMARK 3 REMARK 3 REFINEMENT. REMARK 3  PROGRAM : REFMAC 5.1.24 REMARK 3  AUTHORS : MURSHUDOV, VAGIN, DODSON REMARK 3 REMARK 3   REFINEMENT TARGET: MAXIMUM LIKELIHOOD REMARK 3 REMARK 3  DATA USED IN REFINEMENT. REMARK 3  RESOLUTION RANGE HIGH (ANGSTROMS) :  1.90 REMARK 3  RESOLUTION RANGE LOW (ANGSTROMS) :  19.28 REMARK 3  DATA CUTOFF (SIGMA(F)) : NONE REMARK 3  COMPLETENESS FOR RANGE (%) : 100.00 REMARK 3  NUMBER OF REFLECTIONS :  35284 REMARK 3 REMARK 3  FIT TO DATA USED IN REFINEMENT. REMARK 3  CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3  FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3  R VALUE (WORKING + TEST SET) : 0.18176 REMARK 3  R VALUE (WORKING SET) :  0.17918 REMARK 3  FREE R VALUE :  0.23111 REMARK 3  FREE R VALUE TEST SET SIZE (%) :  5.0 REMARK 3  FREE R VALUE TEST SET COUNT :  1858 REMARK 3 REMARK 3  FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3  TOTAL NUMBER OF BINS USED :    20 REMARK 3  BIN RESOLUTION RANGE HIGH :  1.900 REMARK 3  BIN RESOLUTION RANGE LOW :  1.949 REMARK 3  REFLECTION IN BIN (WORKING SET) :   2544 REMARK 3  BIN R VALUE (WORKING SET) :  0.320 REMARK 3  BIN FREE R VALUE SET COUNT :    134 REMARK 3  BIN FREE R VALUE :  0.358 REMARK 3 REMARK 3  NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3  ALL ATOMS :   2666 REMARK 3 REMARK 3  B VALUES. REMARK 3  FROM WILSON PLOT (A**2) : NULL REMARK 3  MEAN B VALUE (OVERALL, A**2) :  13.186 REMARK 3  OVERALL ANISOTROPIC B VALUE. REMARK 3   B11 (A**2) :    1.01 REMARK 3   B22 (A**2) :   −1.55 REMARK 3   B33 (A**2) :    0.54 REMARK 3   B12 (A**2) :    0.00 REMARK 3   B13 (A**2) :    0.00 REMARK 3   B23 (A**2) :    0.00 REMARK 3 REMARK 3  ESTIMATED OVERALL COORDINATE ERROR. REMARK 3  ESU BASED ON R VALUE (A) :  0.114 REMARK 3  ESU BASED ON FREE R VALUE (A) :  0.123 REMARK 3  ESU BASED ON MAXIMUM LIKELIHOOD (A) :  0.090 REMARK 3  ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2) :  3.289 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3  CORRELATION COEFFICIENT FO-FC :  0.947 REMARK 3  CORRELATION COEFFICIENT FO-FC FREE :  0.916 REMARK 3 REMARK 3  RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT REMARK 3  BOND LENGTHS REFINED ATOMS (A): 2298; 0.029; 0.021 REMARK 3  BOND LENGTHS OTHERS (A): 2000; 0.002; 0.020 REMARK 3  BOND ANGLES REFINED ATOMS (DEGREES): 3096; 3.569; 1.965 REMARK 3  BOND ANGLES OTHERS (DEGREES): 4678; 1.425; 3.000 REMARK 3  TORSION ANGLES, PERIOD 1 (DEGREES):  284; 6.563; 5.000 REMARK 3  CHIRAL-CENTER RESTRAINTS (A**3):  322; 0.442; 0.200 REMARK 3  GENERAL PLANES REFINED ATOMS (A): 2540; 0.021; 0.020 REMARK 3  GENERAL PLANES OTHERS (A):  478; 0.019; 0.020 REMARK 3  NON-BONDED CONTACTS REFINED ATOMS (A):  493; 0.216; 0.300 REMARK 3  NON-BONDED CONTACTS OTHERS (A): 2588; 0.275; 0.300 REMARK 3  NON-BONDED TORSION OTHERS (A): 1402; 0.109; 0.500 REMARK 3  H-BOND (X...Y) REFINED ATOMS (A):  474; 0.292; 0.500 REMARK 3  SYMMETRY VDW REFINED ATOMS (A):  11; 0.190; 0.300 REMARK 3  SYMMETRY VDW OTHERS (A):  59; 0.269; 0.300 REMARK 3  SYMMETRY H-BOND REFINED ATOMS (A):  44; 0.277; 0.500 REMARK 3 REMARK 3  ISOTROPIC THERMAL FACTOR RESTRAINTS. COUNT RMS WEIGHT REMARK 3  MAIN-CHAIN BOND REFINED ATOMS (A**2): 1410; 1.920; 2.000 REMARK 3  MAIN-CHAIN ANGLE REFINED ATOMS (A**2): 2254; 2.794; 3.000 REMARK 3  SIDE-CHAIN BOND REFINED ATOMS (A**2):  888; 2.293; 2.000 REMARK 3  SIDE-CHAIN ANGLE REFINED ATOMS (A**2):  842; 3.356; 3.000 REMARK 3 REMARK 3  NCS RESTRAINTS STATISTICS REMARK 3  NUMBER OF NCS GROUPS: NULL REMARK 3 REMARK 3 REMARK 3  TLS DETAILS REMARK 3  NUMBER OF TLS GROUPS  :   4 REMARK 3 REMARK 3  TLS GROUP  :   1 REMARK 3   NUMBER OF COMPONENTS GROUP  :   1 REMARK 3   COMPONENTS C SSSEQI  TO C SSSEQI REMARK 3   RESIDUE RANGE  : A   2 A   110 REMARK 3   ORIGIN FOR THE GROUP (A):   9.5430  68.2450  26.2250 REMARK 3   T TENSOR REMARK 3   T11:  0.0557 T22:  0.1193 REMARK 3   T33:  0.1386 T12:  0.0268 REMARK 3   T13: −0.0867 T23: −0.0367 REMARK 3   L TENSOR REMARK 3   L11:  2.3675 L22:  1.2064 REMARK 3   L33:  1.0960 L12:  0.8409 REMARK 3   L13:  0.7086 L23:  0.1284 REMARK 3   S TENSOR REMARK 3   S11: −0.1261 S12: −0.1671 S13:  0.2670 REMARK 3   S21:  0.0795 S22: −0.0329 S23:  0.0422 REMARK 3   S31: −0.1451 S32: −0.0618 S33:  0.1590 REMARK 3 REMARK 3  TLS GROUP  :   2 REMARK 3   NUMBER OF COMPONENTS GROUP  :   1 REMARK 3   COMPONENTS C SSSEQI  TO C SSSEQI REMARK 3   RESIDUE RANGE  : A  115 A   144 REMARK 3   ORIGIN FOR THE GROUP (A):  12.2140  42.0860  17.9500 REMARK 3   T TENSOR REMARK 3   T11:  0.0566 T22:  0.1455 REMARK 3   T33:  0.1138 T12:  0.0113 REMARK 3   T13:  0.0248 T23:  0.0103 REMARK 3   L TENSOR REMARK 3   L11:  2.3057 L22:  5.5183 REMARK 3   L33:  2.7772 L12:  1.8588 REMARK 3   L13: −0.9244 L23: −2.0762 REMARK 3   S TENSOR REMARK 3   S11: −0.3277 S12: −0.0677 S13: −0.0462 REMARK 3   S21:  0.0131 S22:  0.1046 S23: −0.3114 REMARK 3   S31:  0.2482 S32:  0.1350 S33:  0.2231 REMARK 3 REMARK 3  TLS GROUP  :   3 REMARK 3   NUMBER OF COMPONENTS GROUP  :   1 REMARK 3   COMPONENTS C SSSEQI  TO C SSSEQI REMARK 3   RESIDUE RANGE  : B   2 B   110 REMARK 3   ORIGIN FOR THE GROUP (A):   8.1830  15.5270  27.3540 REMARK 3   T TENSOR REMARK 3   T11:  0.1252 T22:  0.1062 REMARK 3   T33:  0.0617 T12: −0.0050 REMARK 3   T13:  0.0228 T23:  0.0100 REMARK 3   L TENSOR REMARK 3   L11:  0.5175 L22:  2.2632 REMARK 3   L33:  2.3909 L12: −0.0338 REMARK 3   L13: −0.0735 L23: −1.3985 REMARK 3   S TENSOR REMARK 3   S11:  0.0663 S12: −0.0226 S13: −0.0235 REMARK 3   S21:  0.1096 S22: −0.1191 S23: −0.2038 REMARK 3   S31:  0.1755 S32:  0.0797 S33:  0.0529 REMARK 3 REMARK 3  TLS GROUP  :   4 REMARK 3   NUMBER OF COMPONENTS GROUP  :   1 REMARK 3   COMPONENTS C SSSEQI  TO C SSSEQI REMARK 3   RESIDUE RANGE  : B   115 B   144 REMARK 3   ORIGIN FOR THE GROUP (A):  −1.2080  42.0850  26.2740 REMARK 3   T TENSOR REMARK 3   T11:  0.0583 T22:  0.1343 REMARK 3   T33:  0.0798 T12:  0.0154 REMARK 3   T13:  0.0490 T23: −0.0002 REMARK 3   L TENSOR REMARK 3   L11:  4.7339 L22:  6.5857 REMARK 3   L33:  2.7012 L12: −0.8531 REMARK 3   L13:  1.2150 L23:  0.2365 REMARK 3   S TENSOR REMARK 3   S11: −0.2007 S12: −0.3160 S13:  0.1082 REMARK 3   S21:  0.3828 S22:  0.0444 S23:  0.1488 REMARK 3   S31: −0.1538 S32:  0.0224 S33:  0.1563 REMARK 3 REMARK 3 REMARK 3  BULK SOLVENT MODELLING. REMARK 3   METHOD USED : BABINET MODEL WITH MASK REMARK 3   PARAMETERS FOR MASK CALCULATION REMARK 3   VDW PROBE RADIUS :  1.40 REMARK 3   ION PROBE RADIUS :  0.80 REMARK 3   SHRINKAGE RADIUS :  0.80 REMARK 3 REMARK 3  OTHER REFINEMENT REMARKS: REMARK 3  HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS REMARK 3 CISPEP 1 GLN A  2 VAL A  3 0.00 SSBOND 1 CYS A 117 CYS A 134 SSBOND 2 CYS A 124 CYS A 136 SSBOND 3 CYS A 130 CYS A 142 SSBOND 4 CYS B 117 CYS B 134 SSBOND 5 CYS B 124 CYS B 136 SSBOND 6 CYS B 130 CYS B 142 CRYST1 73.640 216.650 58.160 90.00 90.00 90.00 C 2 2 21 SCALE1 0.013580 0.000000 0.000000 0.00000 SCALE2 0.000000 0.004616 0.000000 0.00000 SCALE3 0.000000 0.000000 0.017194 0.00000 ATOM 1 N GLN A 2 22.714 65.533 17.841 1.00 51.42 N ATOM 3 CA GLN A 2 23.297 65.293 16.498 1.00 52.25 C ATOM 5 CB GLN A 2 23.615 63.798 16.334 1.00 54.95 C ATOM 8 CG GLN A 2 25.085 63.453 16.110 1.00 57.21 C ATOM 11 CD GLN A 2 25.383 62.940 14.662 1.00 59.28 C ATOM 12 OE1 GLN A 2 24.475 62.435 13.953 1.00 59.58 O ATOM 13 NE2 GLN A 2 26.667 63.055 14.238 1.00 59.05 N ATOM 16 C GLN A 2 22.329 65.811 15.433 1.00 48.06 C ATOM 17 O GLN A 2 22.618 65.730 14.219 1.00 49.79 O ATOM 20 N VAL A 3 21.234 66.449 15.853 1.00 41.37 N ATOM 22 CA VAL A 3 20.732 66.626 17.234 1.00 34.29 C ATOM 24 CB VAL A 3 20.931 68.077 17.865 1.00 36.02 C ATOM 26 CG1 VAL A 3 19.746 68.645 18.566 1.00 35.76 C ATOM 30 CG2 VAL A 3 22.043 68.143 18.980 1.00 38.59 C ATOM 34 C VAL A 3 19.240 66.436 16.849 1.00 25.30 C ATOM 35 O VAL A 3 18.798 67.055 15.915 1.00 25.57 O ATOM 36 N ILE A 4 18.489 65.685 17.631 1.00 14.79 N ATOM 38 CA ILE A 4 17.032 65.562 17.436 1.00 9.63 C ATOM 40 CB ILE A 4 16.733 64.041 17.614 1.00 10.11 C ATOM 42 CG1 ILE A 4 17.699 63.151 16.725 1.00 12.96 C ATOM 45 CD1 ILE A 4 17.501 61.602 16.749 1.00 15.54 C ATOM 49 CG2 ILE A 4 15.401 63.628 17.280 1.00 11.19 C ATOM 53 C ILE A 4 16.259 66.398 18.463 1.00 9.80 C ATOM 54 O ILE A 4 16.006 66.065 19.634 1.00 15.16 O ATOM 55 N ASN A 5 15.703 67.414 17.886 1.00 11.17 N ATOM 57 CA ASN A 5 15.339 68.738 18.336 1.00 12.63 C ATOM 59 CB ASN A 5 16.186 69.932 17.409 1.00 11.32 C ATOM 62 CG ASN A 5 16.346 70.863 18.372 1.00 17.03 C ATOM 63 OD1 ASN A 5 16.974 71.871 18.477 1.00 21.67 O ATOM 64 ND2 ASN A 5 15.869 70.160 19.472 1.00 20.49 N ATOM 67 C ASN A 5 13.896 69.048 17.860 1.00 11.85 C ATOM 68 O ASN A 5 13.354 70.061 18.270 1.00 8.88 O ATOM 69 N THR A 6 13.521 68.490 16.731 1.00 11.41 N ATOM 71 CA THR A 6 12.252 68.817 16.131 1.00 12.37 C ATOM 73 CB THR A 6 12.284 68.692 14.624 1.00 14.14 C ATOM 75 OG1 THR A 6 12.544 67.323 14.245 1.00 13.81 O ATOM 77 CG2 THR A 6 13.475 69.517 14.095 1.00 15.31 C ATOM 81 C THR A 6 11.029 68.075 16.682 1.00 12.95 C ATOM 82 O THR A 6 11.163 66.971 17.180 1.00 9.25 O ATOM 83 N PHE A 7 9.844 68.629 16.409 1.00 11.85 N ATOM 85 CA PHE A 7 8.609 68.029 16.838 1.00 11.89 C ATOM 87 CB PHE A 7 7.388 68.783 16.372 1.00 13.02 C ATOM 90 CG PHE A 7 7.092 70.038 17.167 1.00 12.66 C ATOM 91 CD1 PHE A 7 6.661 69.949 18.523 1.00 11.60 C ATOM 93 CE1 PHE A 7 6.522 71.133 19.245 1.00 11.80 C ATOM 95 CZ PHE A 7 6.788 72.375 18.623 1.00 9.66 C ATOM 97 CE2 PHE A 7 7.164 72.444 17.311 1.00 12.14 C ATOM 99 CD2 PHE A 7 7.393 71.295 16.613 1.00 11.78 C ATOM 101 C PHE A 7 8.539 66.601 16.283 1.00 10.82 C ATOM 102 O PHE A 7 8.203 65.682 17.077 1.00 10.87 O ATOM 103 N ASP A 8 8.725 66.458 14.955 1.00 8.74 N ATOM 105 CA ASP A 8 8.624 65.165 14.254 1.00 8.34 C ATOM 107 CB ASP A 8 8.552 65.307 12.761 1.00 8.38 C ATOM 110 CG ASP A 8 7.181 65.848 12.385 1.00 8.22 C ATOM 111 OD1 ASP A 8 6.157 65.350 12.901 1.00 8.55 O ATOM 112 OD2 ASP A 8 7.076 66.792 11.570 1.00 8.62 O ATOM 113 C ASP A 8 9.761 64.248 14.650 1.00 9.81 C ATOM 114 O ASP A 8 9.629 63.060 14.898 1.00 6.21 O ATOM 115 N GLY A 9 10.927 64.821 14.778 1.00 8.66 N ATOM 117 CA GLY A 9 11.972 63.899 15.115 1.00 8.43 C ATOM 120 C GLY A 9 11.842 63.372 16.545 1.00 9.71 C ATOM 121 O GLY A 9 12.204 62.235 16.774 1.00 11.69 O ATOM 122 N VAL A 10 11.526 64.228 17.505 1.00 7.35 N ATOM 124 CA VAL A 10 11.445 63.809 18.896 1.00 6.20 C ATOM 126 CB VAL A 10 11.403 64.969 19.833 1.00 8.73 C ATOM 128 CG1 VAL A 10 11.229 64.568 21.311 1.00 8.03 C ATOM 132 CG2 VAL A 10 12.672 65.808 19.621 1.00 8.63 C ATOM 136 C VAL A 10 10.187 62.955 18.973 1.00 8.44 C ATOM 137 O VAL A 10 10.165 61.921 19.547 1.00 9.75 O ATOM 138 N ALA A 11 9.145 63.249 18.235 1.00 8.06 N ATOM 140 CA ALA A 11 7.956 62.404 18.296 1.00 6.38 C ATOM 142 CB ALA A 11 6.871 63.013 17.484 1.00 7.54 C ATOM 146 C ALA A 11 8.223 60.997 17.795 1.00 8.53 C ATOM 147 O ALA A 11 7.773 59.998 18.394 1.00 8.60 O ATOM 148 N ASP A 12 8.905 60.912 16.653 1.00 10.14 N ATOM 150 CA ASP A 12 9.201 59.571 16.151 1.00 8.94 C ATOM 152 CB ASP A 12 9.841 59.678 14.813 1.00 7.64 C ATOM 155 CG ASP A 12 8.919 60.189 13.703 1.00 10.41 C ATOM 156 OD1 ASP A 12 7.683 60.170 13.869 1.00 12.37 O ATOM 157 OD2 ASP A 12 9.417 60.663 12.675 1.00 9.96 O ATOM 158 C ASP A 12 10.084 58.849 17.163 1.00 9.46 C ATOM 159 O ASP A 12 9.976 57.640 17.320 1.00 8.43 O ATOM 160 N TYR A 13 11.142 59.531 17.599 1.00 9.27 N ATOM 162 CA TYR A 13 12.082 58.969 18.538 1.00 8.06 C ATOM 164 CB TYR A 13 13.273 59.911 18.868 1.00 8.94 C ATOM 167 CG TYR A 13 14.422 59.228 19.526 1.00 7.46 C ATOM 168 CD1 TYR A 13 14.454 58.988 20.878 1.00 7.54 C ATOM 170 CE1 TYR A 13 15.513 58.373 21.475 1.00 7.44 C ATOM 172 CZ TYR A 13 16.661 58.039 20.738 1.00 9.30 C ATOM 173 OH TYR A 13 17.767 57.465 21.340 1.00 12.39 O ATOM 175 CE2 TYR A 13 16.705 58.335 19.414 1.00 9.34 C ATOM 177 CD2 TYR A 13 15.574 58.883 18.787 1.00 7.69 C ATOM 179 C TYR A 13 11.416 58.397 19.783 1.00 11.59 C ATOM 180 O TYR A 13 11.725 57.223 20.139 1.00 11.48 O ATOM 181 N LEU A 14 10.496 59.177 20.357 1.00 9.26 N ATOM 183 CA LEU A 14 9.752 58.756 21.573 1.00 10.40 C ATOM 185 CB LEU A 14 8.804 59.847 22.009 1.00 10.03 C ATOM 188 CG LEU A 14 9.544 60.946 22.748 1.00 10.37 C ATOM 190 CD1 LEU A 14 8.744 62.140 22.732 1.00 10.65 C ATOM 194 CD2 LEU A 14 9.956 60.569 24.104 1.00 9.47 C ATOM 198 C LEU A 14 8.964 57.490 21.225 1.00 9.22 C ATOM 199 O LEU A 14 8.879 56.536 21.962 1.00 8.39 O ATOM 200 N GLN A 15 8.169 57.576 20.148 1.00 6.72 N ATOM 202 CA GLN A 15 7.420 56.409 19.768 1.00 8.38 C ATOM 204 CB GLN A 15 6.320 56.771 18.708 1.00 5.44 C ATOM 207 CG GLN A 15 5.426 57.873 19.269 1.00 7.39 C ATOM 210 CD GLN A 15 4.151 57.906 18.454 1.00 11.03 C ATOM 211 OE1 GLN A 15 4.189 57.833 17.224 1.00 10.64 O ATOM 212 NE2 GLN A 15 3.026 57.918 19.120 1.00 8.47 N ATOM 215 C GLN A 15 8.180 55.150 19.370 1.00 6.72 C ATOM 216 O GLN A 15 7.634 54.043 19.664 1.00 9.19 O ATOM 217 N THR A 16 9.400 55.270 18.868 1.00 6.36 N ATOM 219 CA THR A 16 10.178 54.114 18.658 1.00 6.80 C ATOM 221 CB THR A 16 11.254 54.550 17.647 1.00 9.04 C ATOM 223 OG1 THR A 16 10.557 55.015 16.509 1.00 7.21 O ATOM 225 CG2 THR A 16 12.063 53.377 17.151 1.00 8.26 C ATOM 229 C THR A 16 10.782 53.490 19.924 1.00 7.67 C ATOM 230 O THR A 16 10.765 52.278 20.163 1.00 7.74 O ATOM 231 N TYR A 17 11.577 54.349 20.551 1.00 10.58 N ATOM 233 CA TYR A 17 12.373 53.971 21.706 1.00 8.90 C ATOM 235 CB TYR A 17 13.663 54.748 21.646 1.00 10.32 C ATOM 238 CG TYR A 17 14.368 54.411 20.386 1.00 10.81 C ATOM 239 CD1 TYR A 17 14.456 55.350 19.366 1.00 10.23 C ATOM 241 CE1 TYR A 17 15.098 55.069 18.144 1.00 11.90 C ATOM 243 CZ TYR A 17 15.689 53.811 17.935 1.00 14.28 C ATOM 244 OH TYR A 17 16.254 53.477 16.702 1.00 10.14 O ATOM 246 CE2 TYR A 17 15.619 52.827 18.981 1.00 15.09 C ATOM 248 CD2 TYR A 17 14.874 53.126 20.198 1.00 12.77 C ATOM 250 C TYR A 17 11.812 54.155 23.088 1.00 9.09 C ATOM 251 O TYR A 17 12.393 53.597 23.993 1.00 8.25 O ATOM 252 N HIS A 18 10.744 54.926 23.201 1.00 8.12 N ATOM 254 CA HIS A 18 10.096 55.214 24.508 1.00 10.80 C ATOM 256 CB HIS A 18 9.635 53.948 25.185 1.00 8.03 C ATOM 259 CG HIS A 18 8.345 53.390 24.684 1.00 8.19 C ATOM 260 ND1 HIS A 18 7.755 53.675 23.466 1.00 11.32 N ATOM 262 CE1 HIS A 18 6.644 52.958 23.315 1.00 7.41 C ATOM 264 NE2 HIS A 18 6.516 52.210 24.401 1.00 9.71 N ATOM 266 CD2 HIS A 18 7.598 52.410 25.220 1.00 7.11 C ATOM 268 C HIS A 18 10.945 55.950 25.536 1.00 9.81 C ATOM 269 O HIS A 18 11.005 55.597 26.692 1.00 10.04 O ATOM 270 N LYS A 19 11.799 56.783 25.001 1.00 11.80 N ATOM 272 CA LYS A 19 12.712 57.645 25.737 1.00 12.16 C ATOM 274 CB LYS A 19 14.025 56.952 26.188 1.00 13.25 C ATOM 277 CG LYS A 19 14.911 56.337 25.134 1.00 14.23 C ATOM 280 CD LYS A 19 16.081 55.623 25.742 1.00 18.80 C ATOM 283 CE LYS A 19 16.757 54.993 24.442 1.00 19.86 C ATOM 286 NZ LYS A 19 18.114 54.638 24.964 1.00 21.92 N ATOM 290 C LYS A 19 13.036 58.804 24.789 1.00 14.37 C ATOM 291 O LYS A 19 12.850 58.709 23.596 1.00 12.39 O ATOM 292 N LEU A 20 13.512 59.902 25.369 1.00 13.18 N ATOM 294 CA LEU A 20 13.955 61.071 24.623 1.00 12.39 C ATOM 296 CB LEU A 20 13.959 62.266 25.581 1.00 11.63 C ATOM 299 CG LEU A 20 12.572 62.858 25.874 1.00 12.36 C ATOM 301 CD1 LEU A 20 12.842 63.805 27.067 1.00 14.63 C ATOM 305 CD2 LEU A 20 12.006 63.572 24.678 1.00 12.86 C ATOM 309 C LEU A 20 15.282 60.747 24.083 1.00 8.62 C ATOM 310 O LEU A 20 16.027 59.887 24.673 1.00 11.07 O ATOM 311 N PRO A 21 15.669 61.396 22.978 1.00 7.66 N ATOM 312 CA PRO A 21 17.030 61.185 22.536 1.00 11.67 C ATOM 314 CB PRO A 21 17.153 62.065 21.298 1.00 13.63 C ATOM 317 CG PRO A 21 15.921 62.790 21.172 1.00 12.59 C ATOM 320 CD PRO A 21 14.933 62.257 22.050 1.00 11.31 C ATOM 323 C PRO A 21 18.066 61.784 23.470 1.00 13.18 C ATOM 324 O PRO A 21 17.658 62.525 24.351 1.00 10.88 O ATOM 325 N ASP A 22 19.333 61.502 23.188 1.00 15.40 N ATOM 327 CA ASP A 22 20.443 61.785 24.122 1.00 17.57 C ATOM 329 CB ASP A 22 21.662 61.062 23.593 1.00 24.41 C ATOM 332 CG ASP A 22 21.513 59.510 23.725 1.00 31.16 C ATOM 333 OD1 ASP A 22 20.568 59.028 24.453 1.00 32.54 O ATOM 334 OD2 ASP A 22 22.254 58.691 23.046 1.00 35.54 O ATOM 335 C ASP A 22 20.810 63.259 24.412 1.00 16.73 C ATOM 336 O ASP A 22 21.558 63.592 25.341 1.00 13.24 O ATOM 337 N ASN A 23 20.170 64.169 23.684 1.00 13.14 N ATOM 339 CA ASN A 23 20.316 65.548 23.904 1.00 12.64 C ATOM 341 CB ASN A 23 20.273 66.304 22.595 1.00 13.51 C ATOM 344 CG ASN A 23 18.946 66.065 21.843 1.00 12.48 C ATOM 345 OD1 ASN A 23 18.757 64.931 21.412 1.00 14.44 O ATOM 346 ND2 ASN A 23 18.107 67.103 21.605 1.00 11.53 N ATOM 349 C ASN A 23 19.407 66.138 24.931 1.00 12.26 C ATOM 350 O ASN A 23 19.483 67.323 25.099 1.00 12.57 O ATOM 351 N TYR A 24 18.532 65.358 25.568 1.00 13.29 N ATOM 353 CA TYR A 24 17.668 65.811 26.648 1.00 10.21 C ATOM 355 CB TYR A 24 16.312 65.214 26.465 1.00 10.68 C ATOM 358 CG TYR A 24 15.655 65.918 25.280 1.00 10.21 C ATOM 359 CD1 TYR A 24 14.960 67.105 25.486 1.00 11.90 C ATOM 361 CE1 TYR A 24 14.371 67.769 24.470 1.00 12.10 C ATOM 363 CZ TYR A 24 14.359 67.244 23.188 1.00 9.06 C ATOM 364 OH TYR A 24 13.856 67.954 22.080 1.00 9.96 O ATOM 366 CE2 TYR A 24 14.989 66.015 22.966 1.00 11.14 C ATOM 368 CD2 TYR A 24 15.701 65.379 23.959 1.00 11.66 C ATOM 370 C TYR A 24 18.209 65.569 28.048 1.00 12.21 C ATOM 371 O TYR A 24 18.776 64.512 28.261 1.00 10.15 O ATOM 372 N ILE A 25 18.012 66.559 28.917 1.00 9.70 N ATOM 374 CA ILE A 25 18.206 66.459 30.306 1.00 11.83 C ATOM 376 CB ILE A 25 19.506 67.213 30.728 1.00 11.40 C ATOM 378 CG1 ILE A 25 19.440 68.693 30.416 1.00 9.91 C ATOM 381 CD1 ILE A 25 20.762 69.389 30.853 1.00 10.20 C ATOM 385 CG2 ILE A 25 20.708 66.655 30.035 1.00 11.11 C ATOM 389 C ILE A 25 17.027 67.000 30.988 1.00 11.74 C ATOM 390 O ILE A 25 16.385 67.972 30.528 1.00 8.70 O ATOM 391 N THR A 26 16.734 66.404 32.127 1.00 11.03 N ATOM 393 CA THR A 26 15.652 67.030 32.872 1.00 9.56 C ATOM 395 CB THR A 26 15.130 66.092 33.987 1.00 11.31 C ATOM 397 OG1 THR A 26 16.152 65.889 34.971 1.00 10.67 O ATOM 399 CG2 THR A 26 14.797 64.684 33.576 1.00 12.02 C ATOM 403 C THR A 26 15.942 68.370 33.479 1.00 11.08 C ATOM 404 O THR A 26 17.095 68.793 33.616 1.00 14.78 O ATOM 405 N LYS A 27 14.906 68.974 34.062 1.00 13.18 N ATOM 407 CA LYS A 27 15.031 70.284 34.711 1.00 15.98 C ATOM 409 CB LYS A 27 13.708 70.925 35.272 1.00 17.62 C ATOM 412 CG LYS A 27 12.763 71.828 34.349 1.00 25.05 C ATOM 415 CD LYS A 27 11.112 71.441 34.443 1.00 26.12 C ATOM 418 CE LYS A 27 10.193 71.244 33.189 1.00 27.01 C ATOM 421 NZ LYS A 27 9.557 72.396 32.193 1.00 26.80 N ATOM 425 C LYS A 27 16.068 70.157 35.809 1.00 13.27 C ATOM 426 O LYS A 27 16.999 70.916 35.857 1.00 14.27 O ATOM 427 N SER A 28 15.877 69.187 36.707 1.00 9.50 N ATOM 429 CA SER A 28 16.734 68.983 37.841 1.00 10.56 C ATOM 431 CB SER A 28 16.099 67.834 38.613 1.00 11.46 C ATOM 434 OG SER A 28 15.088 68.316 39.454 1.00 9.88 O ATOM 436 C SER A 28 18.135 68.602 37.415 1.00 9.36 C ATOM 437 O SER A 28 19.085 69.048 38.030 1.00 6.96 O ATOM 438 N GLU A 29 18.255 67.802 36.373 1.00 11.20 N ATOM 440 CA GLU A 29 19.645 67.503 35.960 1.00 12.45 C ATOM 442 CB GLU A 29 19.714 66.495 34.796 1.00 14.00 C ATOM 445 CG GLU A 29 19.473 65.002 35.018 1.00 19.06 C ATOM 448 CD GLU A 29 19.686 64.358 33.618 1.00 21.71 C ATOM 449 OE1 GLU A 29 20.911 64.341 33.214 1.00 25.61 O ATOM 450 OE2 GLU A 29 18.719 64.383 32.868 1.00 15.51 O ATOM 451 C GLU A 29 20.303 68.763 35.454 1.00 8.44 C ATOM 452 O GLU A 29 21.504 69.019 35.698 1.00 10.70 O ATOM 453 N ALA A 30 19.563 69.564 34.720 1.00 7.23 N ATOM 455 CA ALA A 30 20.122 70.807 34.135 1.00 7.75 C ATOM 457 CB ALA A 30 19.039 71.472 33.362 1.00 9.55 C ATOM 461 C ALA A 30 20.596 71.777 35.251 1.00 8.76 C ATOM 462 O ALA A 30 21.699 72.300 35.197 1.00 11.39 O ATOM 463 N GLN A 31 19.833 71.804 36.339 1.00 9.29 N ATOM 465 CA GLN A 31 20.103 72.676 37.441 1.00 11.25 C ATOM 467 CB GLN A 31 18.966 72.692 38.463 1.00 11.04 C ATOM 470 CG GLN A 31 17.718 73.425 37.952 1.00 14.12 C ATOM 473 CD GLN A 31 16.494 73.253 38.898 1.00 19.64 C ATOM 474 OE1 GLN A 31 16.506 72.345 39.712 1.00 23.07 O ATOM 475 NE2 GLN A 31 15.471 74.101 38.819 1.00 20.34 N ATOM 478 C GLN A 31 21.429 72.201 38.077 1.00 9.02 C ATOM 479 O GLN A 31 22.183 73.029 38.506 1.00 11.78 O ATOM 480 N ALA A 32 21.561 70.901 38.279 1.00 10.18 N ATOM 482 CA ALA A 32 22.677 70.236 38.989 1.00 11.47 C ATOM 484 CB ALA A 32 22.481 68.753 39.034 1.00 12.44 C ATOM 488 C ALA A 32 23.870 70.518 38.116 1.00 13.28 C ATOM 489 O ALA A 32 24.914 70.888 38.646 1.00 11.93 O ATOM 490 N LEU A 33 23.662 70.518 36.792 1.00 13.42 N ATOM 492 CA LEU A 33 24.793 70.844 35.892 1.00 14.02 C ATOM 494 CB LEU A 33 24.534 70.318 34.443 1.00 15.03 C ATOM 497 CG LEU A 33 24.448 68.788 34.388 1.00 16.07 C ATOM 499 CD1 LEU A 33 23.979 68.378 32.994 1.00 18.21 C ATOM 503 CD2 LEU A 33 25.736 68.108 34.800 1.00 18.74 C ATOM 507 C LEU A 33 25.104 72.313 35.800 1.00 11.26 C ATOM 508 O LEU A 33 26.052 72.707 35.094 1.00 14.47 O ATOM 509 N GLY A 34 24.303 73.160 36.434 1.00 10.53 N ATOM 511 CA GLY A 34 24.621 74.592 36.558 1.00 9.48 C ATOM 514 C GLY A 34 23.644 75.540 35.935 1.00 9.94 C ATOM 515 O GLY A 34 23.762 76.720 36.109 1.00 11.20 O ATOM 516 N TRP A 35 22.552 75.045 35.343 1.00 8.51 N ATOM 518 CA TRP A 35 21.516 75.886 34.712 1.00 10.37 C ATOM 520 CB TRP A 35 20.516 75.006 33.978 1.00 9.49 C ATOM 523 CG TRP A 35 19.403 75.797 33.464 1.00 10.46 C ATOM 524 CD1 TRP A 35 19.413 76.830 32.588 1.00 10.67 C ATOM 526 NE1 TRP A 35 18.126 77.221 32.338 1.00 10.69 N ATOM 528 CE2 TRP A 35 17.276 76.364 32.953 1.00 10.95 C ATOM 529 CD2 TRP A 35 18.055 75.494 33.714 1.00 11.03 C ATOM 530 CE3 TRP A 35 17.421 74.517 34.474 1.00 12.02 C ATOM 532 CZ3 TRP A 35 16.043 74.477 34.433 1.00 16.19 C ATOM 534 CH2 TRP A 35 15.298 75.423 33.759 1.00 14.45 C ATOM 536 CZ2 TRP A 35 15.912 76.386 33.026 1.00 13.57 C ATOM 538 C TRP A 35 20.759 76.563 35.769 1.00 11.80 C ATOM 539 O TRP A 35 20.480 75.978 36.787 1.00 13.46 O ATOM 540 N VAL A 36 20.642 77.854 35.568 1.00 13.51 N ATOM 542 CA VAL A 36 19.850 78.721 36.411 1.00 14.52 C ATOM 544 CB VAL A 36 20.709 79.850 37.056 1.00 14.17 C ATOM 546 CG1 VAL A 36 19.720 80.804 37.721 1.00 14.44 C ATOM 550 CG2 VAL A 36 21.728 79.251 37.936 1.00 15.02 C ATOM 554 C VAL A 36 18.845 79.361 35.471 1.00 14.86 C ATOM 555 O VAL A 36 19.280 80.072 34.565 1.00 14.15 O ATOM 556 N ALA A 37 17.552 79.152 35.681 1.00 16.80 N ATOM 558 CA ALA A 37 16.602 79.612 34.642 1.00 19.03 C ATOM 560 CB ALA A 37 15.185 79.302 35.065 1.00 19.86 C ATOM 564 C ALA A 37 16.761 81.111 34.322 1.00 21.10 C ATOM 565 O ALA A 37 16.893 81.598 33.201 1.00 22.32 O ATOM 566 N SER A 38 16.864 81.921 35.342 1.00 21.48 N ATOM 568 CA SER A 38 16.846 83.354 35.069 1.00 21.38 C ATOM 570 CB SER A 38 16.521 84.067 36.372 1.00 19.41 C ATOM 573 OG SER A 38 17.599 83.864 37.277 1.00 17.79 O ATOM 575 C SER A 38 18.165 83.861 34.440 1.00 22.75 C ATOM 576 O SER A 38 18.301 85.070 34.290 1.00 24.55 O ATOM 577 N LYS A 39 19.210 83.047 34.266 1.00 21.17 N ATOM 579 CA LYS A 39 20.406 83.423 33.529 1.00 21.64 C ATOM 581 CB LYS A 39 21.725 82.845 34.110 1.00 24.13 C ATOM 584 CG LYS A 39 22.192 83.242 35.464 1.00 26.02 C ATOM 587 CD LYS A 39 23.733 83.693 35.579 1.00 28.80 C ATOM 590 CE LYS A 39 24.843 82.596 35.622 1.00 29.09 C ATOM 593 NZ LYS A 39 26.213 83.289 35.676 1.00 27.36 N ATOM 597 C LYS A 39 20.403 83.022 32.048 1.00 19.45 C ATOM 598 O LYS A 39 21.295 83.409 31.286 1.00 20.46 O ATOM 599 N GLY A 40 19.470 82.199 31.602 1.00 15.53 N ATOM 601 CA GLY A 40 19.449 81.892 30.173 1.00 16.20 C ATOM 604 C GLY A 40 20.691 81.083 29.799 1.00 14.65 C ATOM 605 O GLY A 40 21.128 81.158 28.663 1.00 14.14 O ATOM 606 N ASN A 41 21.246 80.304 30.737 1.00 13.48 N ATOM 608 CA ASN A 41 22.602 79.734 30.524 1.00 12.48 C ATOM 610 CB ASN A 41 23.431 80.019 31.766 1.00 11.98 C ATOM 613 CG ASN A 41 23.007 79.186 33.011 1.00 12.56 C ATOM 614 OD1 ASN A 41 21.840 78.779 33.155 1.00 10.26 O ATOM 615 ND2 ASN A 41 23.956 79.002 33.944 1.00 9.96 N ATOM 618 C ASN A 41 22.632 78.213 30.191 1.00 11.89 C ATOM 619 O ASN A 41 23.673 77.509 30.256 1.00 14.37 O ATOM 620 N LEU A 42 21.514 77.703 29.710 1.00 12.36 N ATOM 622 CA LEU A 42 21.402 76.226 29.582 1.00 12.49 C ATOM 624 CB LEU A 42 20.084 75.745 29.009 1.00 10.42 C ATOM 627 CG LEU A 42 19.937 74.236 28.890 1.00 11.23 C ATOM 629 CD1 LEU A 42 20.038 73.626 30.265 1.00 12.26 C ATOM 633 CD2 LEU A 42 18.518 74.008 28.264 1.00 11.52 C ATOM 637 C LEU A 42 22.519 75.702 28.653 1.00 11.43 C ATOM 638 O LEU A 42 23.242 74.740 28.955 1.00 9.65 O ATOM 639 N ALA A 43 22.647 76.369 27.522 1.00 10.58 N ATOM 641 CA ALA A 43 23.583 75.901 26.544 1.00 10.42 C ATOM 643 CB ALA A 43 23.235 76.524 25.214 1.00 12.50 C ATOM 647 C ALA A 43 25.004 76.298 26.884 1.00 12.63 C ATOM 648 O ALA A 43 25.954 75.777 26.273 1.00 10.33 O ATOM 649 N ASP A 44 25.173 77.151 27.882 1.00 11.19 N ATOM 651 CA ASP A 44 26.547 77.343 28.254 1.00 12.65 C ATOM 653 CB ASP A 44 26.698 78.658 28.997 1.00 13.47 C ATOM 656 CG ASP A 44 26.281 79.841 28.157 1.00 15.50 C ATOM 657 OD1 ASP A 44 26.671 79.946 26.990 1.00 13.20 O ATOM 658 OD2 ASP A 44 25.532 80.690 28.634 1.00 19.68 O ATOM 659 C ASP A 44 26.966 76.197 29.189 1.00 13.99 C ATOM 660 O ASP A 44 28.062 75.754 29.121 1.00 11.77 O ATOM 661 N VAL A 45 26.076 75.730 30.064 1.00 12.97 N ATOM 663 CA VAL A 45 26.468 74.688 30.983 1.00 8.34 C ATOM 665 CB VAL A 45 25.779 74.756 32.343 1.00 9.15 C ATOM 667 CG1 VAL A 45 26.054 76.076 33.087 1.00 11.49 C ATOM 671 CG2 VAL A 45 24.267 74.554 32.247 1.00 7.95 C ATOM 675 C VAL A 45 26.299 73.328 30.373 1.00 8.80 C ATOM 676 O VAL A 45 26.934 72.410 30.809 1.00 8.81 O ATOM 677 N ALA A 46 25.474 73.205 29.359 1.00 8.48 N ATOM 679 CA ALA A 46 25.183 71.921 28.744 1.00 10.55 C ATOM 681 CB ALA A 46 23.884 71.323 29.281 1.00 9.59 C ATOM 685 C ALA A 46 25.117 72.035 27.257 1.00 9.69 C ATOM 686 O ALA A 46 24.005 72.015 26.706 1.00 8.90 O ATOM 687 N PRO A 47 26.247 72.282 26.617 1.00 11.83 N ATOM 688 CA PRO A 47 26.241 72.537 25.155 1.00 12.60 C ATOM 690 CB PRO A 47 27.722 72.459 24.751 1.00 12.43 C ATOM 693 CG PRO A 47 28.426 73.045 25.923 1.00 12.70 C ATOM 696 CD PRO A 47 27.605 72.438 27.159 1.00 12.26 C ATOM 699 C PRO A 47 25.458 71.487 24.393 1.00 11.89 C ATOM 700 O PRO A 47 25.723 70.339 24.669 1.00 14.14 O ATOM 701 N GLY A 48 24.579 71.821 23.457 1.00 11.22 N ATOM 703 CA GLY A 48 23.812 70.835 22.738 1.00 12.82 C ATOM 706 C GLY A 48 22.614 70.235 23.430 1.00 13.24 C ATOM 707 O GLY A 48 21.888 69.530 22.788 1.00 9.40 O ATOM 708 N LYS A 49 22.376 70.531 24.694 1.00 13.30 N ATOM 710 CA LYS A 49 21.263 69.942 25.439 1.00 11.44 C ATOM 712 CB LYS A 49 21.695 69.461 26.844 1.00 10.85 C ATOM 715 CG LYS A 49 22.941 68.549 26.822 1.00 11.14 C ATOM 718 CD LYS A 49 22.764 67.266 26.000 1.00 13.18 C ATOM 721 CE LYS A 49 24.006 66.318 26.094 1.00 15.17 C ATOM 724 NZ LYS A 49 23.634 64.958 25.791 1.00 15.55 N ATOM 728 C LYS A 49 20.044 70.839 25.492 1.00 12.76 C ATOM 729 O LYS A 49 20.112 72.030 25.213 1.00 14.86 O ATOM 730 N SER A 50 18.913 70.163 25.630 1.00 10.73 N ATOM 732 CA SER A 50 17.619 70.741 25.875 1.00 7.36 C ATOM 734 CB SER A 50 16.736 70.450 24.697 1.00 8.47 C ATOM 737 OG SER A 50 17.234 70.891 23.425 1.00 8.66 O ATOM 739 C SER A 50 17.026 70.165 27.140 1.00 11.33 C ATOM 740 O SER A 50 17.261 68.990 27.459 1.00 13.01 O ATOM 741 N ILE A 51 16.097 70.924 27.762 1.00 11.26 N ATOM 743 CA ILE A 51 15.379 70.391 28.867 1.00 10.46 C ATOM 745 CB ILE A 51 14.534 71.580 29.537 1.00 13.60 C ATOM 747 CG1 ILE A 51 15.343 72.572 30.368 1.00 14.63 C ATOM 750 CD1 ILE A 51 16.482 72.131 30.836 1.00 14.11 C ATOM 754 CG2 ILE A 51 13.746 70.994 30.651 1.00 13.76 C ATOM 758 C ILE A 51 14.324 69.432 28.370 1.00 9.40 C ATOM 759 O ILE A 51 13.584 69.744 27.406 1.00 10.07 O ATOM 760 N GLY A 52 14.145 68.308 29.068 1.00 7.01 N ATOM 762 CA GLY A 52 13.082 67.410 28.665 1.00 11.07 C ATOM 765 C GLY A 52 13.076 66.192 29.552 1.00 9.40 C ATOM 766 O GLY A 52 13.998 65.806 30.274 1.00 10.01 O ATOM 767 N GLY A 53 11.854 65.731 29.690 1.00 7.49 N ATOM 769 CA GLY A 53 11.526 64.546 30.398 1.00 7.92 C ATOM 772 C GLY A 53 10.708 64.622 31.623 1.00 9.77 C ATOM 773 O GLY A 53 10.445 63.577 32.196 1.00 10.62 O ATOM 774 N ASP A 54 10.367 65.820 32.046 1.00 11.61 N ATOM 776 CA ASP A 54 9.672 65.998 33.300 1.00 9.49 C ATOM 778 CB ASP A 54 9.877 67.375 33.845 1.00 11.50 C ATOM 781 CG ASP A 54 11.409 67.642 34.138 1.00 14.34 C ATOM 782 OD1 ASP A 54 11.970 66.760 34.820 1.00 16.87 O ATOM 783 OD2 ASP A 54 12.053 68.563 33.625 1.00 9.69 O ATOM 784 C ASP A 54 8.205 65.732 33.154 1.00 10.32 C ATOM 785 O ASP A 54 7.671 65.938 32.090 1.00 8.97 O ATOM 786 N ILE A 55 7.556 65.472 34.266 1.00 9.81 N ATOM 788 CA ILE A 55 6.112 65.406 34.259 1.00 12.28 C ATOM 790 CB ILE A 55 5.632 64.968 35.646 1.00 11.93 C ATOM 792 CG1 ILE A 55 5.794 63.507 35.611 1.00 15.09 C ATOM 795 CD1 ILE A 55 6.656 63.222 36.568 1.00 19.15 C ATOM 799 CG2 ILE A 55 4.080 65.112 35.721 1.00 13.77 C ATOM 803 C ILE A 55 5.456 66.735 33.987 1.00 12.40 C ATOM 804 O ILE A 55 5.821 67.753 34.489 1.00 12.96 O ATOM 805 N PHE A 56 4.494 66.722 33.104 1.00 12.87 N ATOM 807 CA PHE A 56 3.594 67.817 32.876 1.00 14.48 C ATOM 809 CB PHE A 56 3.292 67.952 31.372 1.00 15.22 C ATOM 812 CG PHE A 56 2.394 69.048 31.061 1.00 13.94 C ATOM 813 CD1 PHE A 56 2.756 70.358 31.243 1.00 14.54 C ATOM 815 CE1 PHE A 56 1.835 71.387 30.916 1.00 14.29 C ATOM 817 CZ PHE A 56 0.550 71.078 30.557 1.00 15.46 C ATOM 819 CE2 PHE A 56 0.226 69.794 30.283 1.00 14.32 C ATOM 821 CD2 PHE A 56 1.111 68.779 30.589 1.00 17.12 C ATOM 823 C PHE A 56 2.315 67.389 33.561 1.00 14.02 C ATOM 824 O PHE A 56 1.619 66.341 33.309 1.00 14.15 O ATOM 825 N SER A 57 1.999 68.276 34.478 1.00 14.80 N ATOM 827 CA SER A 57 0.886 68.019 35.361 1.00 16.86 C ATOM 829 CB SER A 57 1.077 68.915 36.606 1.00 19.01 C ATOM 832 OG SER A 57 0.349 70.078 36.232 1.00 23.78 O ATOM 834 C SER A 57 −0.490 68.175 34.686 1.00 14.80 C ATOM 835 O SER A 57 −1.423 67.551 35.126 1.00 13.55 O ATOM 836 N ASN A 58 −0.639 68.880 33.566 1.00 14.70 N ATOM 838 CA ASN A 58 −1.909 68.934 32.798 1.00 13.86 C ATOM 840 CB ASN A 58 −2.305 67.579 32.253 1.00 12.61 C ATOM 843 CG ASN A 58 −3.415 67.687 31.230 1.00 12.21 C ATOM 844 OD1 ASN A 58 −3.457 68.669 30.508 1.00 14.42 O ATOM 845 ND2 ASN A 58 −4.234 66.664 31.077 1.00 10.60 N ATOM 848 C ASN A 58 −3.061 69.513 33.640 1.00 16.38 C ATOM 849 O ASN A 58 −4.238 69.071 33.529 1.00 15.38 O ATOM 850 N ARG A 59 −2.648 70.538 34.401 1.00 18.42 N ATOM 852 CA ARG A 59 −3.475 71.224 35.413 1.00 23.61 C ATOM 854 CB ARG A 59 −2.735 72.444 36.041 1.00 26.92 C ATOM 857 CG ARG A 59 −2.301 72.317 37.549 1.00 32.22 C ATOM 860 CD ARG A 59 −1.041 73.159 38.035 1.00 35.70 C ATOM 863 NE ARG A 59 0.042 72.323 38.633 1.00 39.31 N ATOM 865 CZ ARG A 59 1.377 72.328 38.378 1.00 41.80 C ATOM 866 NH1 ARG A 59 1.975 73.090 37.445 1.00 42.97 N ATOM 869 NH2 ARG A 59 2.145 71.456 39.033 1.00 41.96 N ATOM 872 C ARG A 59 −4.808 71.680 34.787 1.00 20.35 C ATOM 873 O ARG A 59 −5.877 71.410 35.299 1.00 24.33 O ATOM 874 N GLU A 60 −4.731 72.368 33.665 1.00 19.25 N ATOM 876 CA GLU A 60 −5.905 72.954 32.977 1.00 20.37 C ATOM 878 CB GLU A 60 −5.385 74.077 32.036 1.00 23.39 C ATOM 881 CG GLU A 60 −4.458 75.170 32.640 1.00 26.33 C ATOM 884 CD GLU A 60 −4.214 76.380 31.668 1.00 30.05 C ATOM 885 OE1 GLU A 60 −5.095 77.283 31.635 1.00 31.61 O ATOM 886 OE2 GLU A 60 −3.236 76.514 30.835 1.00 30.87 O ATOM 887 C GLU A 60 −6.709 71.902 32.209 1.00 16.43 C ATOM 888 O GLU A 60 −7.791 72.180 31.655 1.00 17.87 O ATOM 889 N GLY A 61 −6.229 70.661 32.169 1.00 15.60 N ATOM 891 CA GLY A 61 −6.946 69.555 31.558 1.00 14.04 C ATOM 894 C GLY A 61 −7.052 69.738 30.057 1.00 13.71 C ATOM 895 O GLY A 61 −7.874 69.132 29.405 1.00 11.69 O ATOM 896 N LYS A 62 −6.209 70.566 29.473 1.00 14.43 N ATOM 898 CA LYS A 62 −6.166 70.684 28.018 1.00 15.74 C ATOM 900 CB LYS A 62 −5.296 71.872 27.609 1.00 16.93 C ATOM 903 CG LYS A 62 −5.987 73.225 27.903 1.00 18.53 C ATOM 906 CD LYS A 62 −5.027 74.380 28.045 1.00 18.80 C ATOM 909 CE LYS A 62 −5.824 75.691 28.420 1.00 19.77 C ATOM 912 NZ LYS A 62 −4.845 76.708 28.828 1.00 20.13 N ATOM 916 C LYS A 62 −5.702 69.470 27.267 1.00 16.16 C ATOM 917 O LYS A 62 −6.122 69.221 26.138 1.00 17.96 O ATOM 918 N LEU A 63 −4.817 68.680 27.844 1.00 15.98 N ATOM 920 CA LEU A 63 −4.536 67.419 27.210 1.00 14.10 C ATOM 922 CB LEU A 63 −3.202 67.029 27.637 1.00 15.14 C ATOM 925 CG LEU A 63 −2.182 67.945 27.006 1.00 18.20 C ATOM 927 CD1 LEU A 63 −0.936 67.468 27.771 1.00 17.41 C ATOM 931 CD2 LEU A 63 −1.957 67.758 25.449 1.00 18.52 C ATOM 935 C LEU A 63 −5.494 66.341 27.686 1.00 15.09 C ATOM 936 O LEU A 63 −5.976 66.439 28.819 1.00 11.32 O ATOM 937 N PRO A 64 −5.941 65.420 26.829 1.00 12.99 N ATOM 938 CA PRO A 64 −6.831 64.396 27.386 1.00 15.31 C ATOM 940 CB PRO A 64 −7.160 63.478 26.232 1.00 14.87 C ATOM 943 CG PRO A 64 −6.007 63.679 25.152 1.00 15.10 C ATOM 946 CD PRO A 64 −5.456 65.079 25.495 1.00 14.91 C ATOM 949 C PRO A 64 −6.345 63.488 28.528 1.00 15.62 C ATOM 950 O PRO A 64 −5.276 62.915 28.427 1.00 15.46 O ATOM 951 N GLY A 65 −7.239 63.267 29.472 1.00 17.38 N ATOM 953 CA GLY A 65 −6.995 62.584 30.724 1.00 19.51 C ATOM 956 C GLY A 65 −7.455 61.157 30.634 1.00 19.61 C ATOM 957 O GLY A 65 −8.458 60.920 29.978 1.00 18.11 O ATOM 958 N LYS A 66 −6.798 60.211 31.308 1.00 21.09 N ATOM 960 CA LYS A 66 −7.378 58.848 31.254 1.00 23.49 C ATOM 962 CB LYS A 66 −6.770 57.952 30.182 1.00 25.72 C ATOM 965 CG LYS A 66 −7.570 56.786 29.647 1.00 28.00 C ATOM 968 CD LYS A 66 −6.737 55.758 28.714 1.00 29.97 C ATOM 971 CE LYS A 66 −7.418 54.358 28.638 1.00 30.91 C ATOM 974 NZ LYS A 66 −7.560 53.792 27.285 1.00 31.32 N ATOM 978 C LYS A 66 −6.901 58.312 32.524 1.00 23.01 C ATOM 979 O LYS A 66 −5.834 58.738 32.962 1.00 22.45 O ATOM 980 N SER A 67 −7.622 57.331 33.061 1.00 21.46 N ATOM 982 CA SER A 67 −7.136 56.739 34.323 1.00 21.32 C ATOM 984 CB SER A 67 −8.240 55.791 34.802 1.00 24.97 C ATOM 987 OG SER A 67 −7.681 55.050 35.861 1.00 27.07 O ATOM 989 C SER A 67 −5.787 55.980 34.098 1.00 18.02 C ATOM 990 O SER A 67 −5.592 55.247 33.116 1.00 19.08 O ATOM 991 N GLY A 68 −4.800 56.276 34.899 1.00 14.53 N ATOM 993 CA GLY A 68 −3.446 55.766 34.748 1.00 14.52 C ATOM 996 C GLY A 68 −2.518 56.476 33.766 1.00 11.70 C ATOM 997 O GLY A 68 −1.377 56.160 33.668 1.00 11.23 O ATOM 998 N ARG A 69 −2.997 57.470 33.062 1.00 12.48 N ATOM 1000 CA ARG A 69 −2.153 58.194 32.121 1.00 12.39 C ATOM 1002 CB ARG A 69 −3.080 58.611 30.974 1.00 10.55 C ATOM 1005 CG ARG A 69 −2.345 59.529 30.015 1.00 12.38 C ATOM 1008 CD ARG A 69 −3.161 60.232 28.957 1.00 13.48 C ATOM 1011 NE ARG A 69 −3.600 59.225 27.992 1.00 13.13 N ATOM 1013 CZ ARG A 69 −4.757 59.251 27.381 1.00 15.18 C ATOM 1014 NH1 ARG A 69 −5.675 60.230 27.544 1.00 13.45 N ATOM 1017 NH2 ARG A 69 −5.086 58.182 26.639 1.00 15.98 N ATOM 1020 C ARG A 69 −1.486 59.400 32.714 1.00 10.39 C ATOM 1021 O ARG A 69 −2.146 60.226 33.368 1.00 13.47 O ATOM 1022 N THR A 70 −0.184 59.494 32.567 1.00 9.24 N ATOM 1024 CA THR A 70 0.531 60.610 32.964 1.00 10.73 C ATOM 1026 CB THR A 70 1.641 60.202 33.945 1.00 15.19 C ATOM 1028 OG1 THR A 70 2.094 58.921 33.584 1.00 18.14 O ATOM 1030 CG2 THR A 70 1.016 59.865 35.338 1.00 17.68 C ATOM 1034 C THR A 70 1.091 61.251 31.715 1.00 11.07 C ATOM 1035 O THR A 70 1.206 60.564 30.704 1.00 9.88 O ATOM 1036 N TRP A 71 1.639 62.466 31.834 1.00 9.15 N ATOM 1038 CA TRP A 71 2.141 63.188 30.673 1.00 9.48 C ATOM 1040 CB TRP A 71 1.238 64.391 30.336 1.00 9.88 C ATOM 1043 CG TRP A 71 −0.079 64.100 29.723 1.00 10.12 C ATOM 1044 CD1 TRP A 71 −1.244 63.942 30.410 1.00 9.86 C ATOM 1046 NE1 TRP A 71 −2.246 63.682 29.513 1.00 13.26 N ATOM 1048 CE2 TRP A 71 −1.707 63.641 28.245 1.00 10.61 C ATOM 1049 CD2 TRP A 71 −0.379 63.953 28.342 1.00 8.98 C ATOM 1050 CE3 TRP A 71 0.398 63.946 27.152 1.00 10.37 C ATOM 1052 CZ3 TRP A 71 −0.162 63.667 25.981 1.00 8.20 C ATOM 1054 CH2 TRP A 71 −1.485 63.405 25.876 1.00 10.45 C ATOM 1056 CZ2 TRP A 71 −2.293 63.358 27.006 1.00 11.14 C ATOM 1058 C TRP A 71 3.527 63.726 31.033 1.00 10.14 C ATOM 1059 O TRP A 71 3.738 64.165 32.188 1.00 8.24 O ATOM 1060 N ARG A 72 4.385 63.889 30.029 1.00 8.38 N ATOM 1062 CA ARG A 72 5.687 64.373 30.238 1.00 9.32 C ATOM 1064 CB ARG A 72 6.717 63.216 30.167 1.00 11.31 C ATOM 1067 CG ARG A 72 6.993 62.457 31.497 1.00 13.27 C ATOM 1070 CD ARG A 72 7.894 61.261 31.445 1.00 13.70 C ATOM 1073 NE ARG A 72 7.765 60.444 32.612 1.00 16.16 N ATOM 1075 CZ ARG A 72 8.301 60.753 33.805 1.00 20.44 C ATOM 1076 NH1 ARG A 72 9.077 61.831 33.985 1.00 17.83 N ATOM 1079 NH2 ARG A 72 8.008 60.008 34.861 1.00 21.24 N ATOM 1082 C ARG A 72 5.905 65.353 29.118 1.00 10.57 C ATOM 1083 O ARG A 72 5.276 65.342 28.063 1.00 12.04 O ATOM 1084 N GLU A 73 6.918 66.176 29.292 1.00 12.14 N ATOM 1086 CA GLU A 73 7.159 67.189 28.257 1.00 12.34 C ATOM 1088 CB GLU A 73 6.705 68.554 28.815 1.00 13.21 C ATOM 1091 CG GLU A 73 7.474 68.977 30.056 1.00 11.26 C ATOM 1094 CD GLU A 73 7.316 70.435 30.333 1.00 12.31 C ATOM 1095 OE1 GLU A 73 7.794 71.274 29.566 1.00 14.70 O ATOM 1096 OE2 GLU A 73 6.628 70.788 31.316 1.00 17.26 O ATOM 1097 C GLU A 73 8.621 67.330 27.931 1.00 11.97 C ATOM 1098 O GLU A 73 9.498 66.851 28.673 1.00 10.83 O ATOM 1099 N ALA A 74 8.920 67.876 26.764 1.00 11.65 N ATOM 1101 CA ALA A 74 10.303 68.128 26.423 1.00 9.14 C ATOM 1103 CB ALA A 74 10.934 67.030 25.740 1.00 7.95 C ATOM 1107 C ALA A 74 10.313 69.384 25.559 1.00 10.16 C ATOM 1108 O ALA A 74 9.341 69.674 24.900 1.00 13.65 O ATOM 1109 N ASP A 75 11.402 70.174 25.667 1.00 9.31 N ATOM 1111 CA ASP A 75 11.577 71.351 24.833 1.00 8.31 C ATOM 1113 CB ASP A 75 12.737 72.217 25.364 1.00 8.64 C ATOM 1116 CG ASP A 75 12.404 72.980 26.649 1.00 9.50 C ATOM 1117 OD1 ASP A 75 11.471 72.519 27.347 1.00 11.90 O ATOM 1118 OD2 ASP A 75 13.015 74.057 26.968 1.00 14.38 O ATOM 1119 C ASP A 75 11.904 70.993 23.414 1.00 7.79 C ATOM 1120 O ASP A 75 12.755 70.147 23.183 1.00 8.59 O ATOM 1121 N ILE A 76 11.279 71.673 22.447 1.00 11.70 N ATOM 1123 CA ILE A 76 11.618 71.533 21.068 1.00 11.46 C ATOM 1125 CB ILE A 76 10.285 71.222 20.407 1.00 12.87 C ATOM 1127 CG1 ILE A 76 9.829 69.860 20.914 1.00 13.71 C ATOM 1130 CD1 ILE A 76 10.590 68.697 20.238 1.00 11.84 C ATOM 1134 CG2 ILE A 76 10.407 71.190 18.939 1.00 12.63 C ATOM 1138 C ILE A 76 12.130 72.855 20.595 1.00 11.92 C ATOM 1139 O ILE A 76 11.713 73.953 21.039 1.00 11.71 O ATOM 1140 N ASN A 77 12.929 72.701 19.563 1.00 10.75 N ATOM 1142 CA ASN A 77 13.606 73.727 18.759 1.00 11.46 C ATOM 1144 CB ASN A 77 12.614 74.609 18.043 1.00 12.26 C ATOM 1147 CG ASN A 77 11.806 73.898 17.003 1.00 11.68 C ATOM 1148 OD1 ASN A 77 12.299 73.103 16.183 1.00 12.28 O ATOM 1149 ND2 ASN A 77 10.549 74.329 16.919 1.00 11.36 N ATOM 1152 C ASN A 77 14.607 74.614 19.557 1.00 11.79 C ATOM 1153 O ASN A 77 14.834 75.788 19.262 1.00 15.08 O ATOM 1154 N TYR A 78 15.059 74.083 20.655 1.00 8.82 N ATOM 1156 CA TYR A 78 15.853 74.919 21.572 1.00 12.79 C ATOM 1158 CB TYR A 78 15.783 74.407 23.024 1.00 11.72 C ATOM 1161 CG TYR A 78 16.794 75.115 23.878 1.00 12.22 C ATOM 1162 CD1 TYR A 78 16.509 76.414 24.352 1.00 12.36 C ATOM 1164 CE1 TYR A 78 17.467 77.153 25.060 1.00 9.98 C ATOM 1166 CZ TYR A 78 18.672 76.533 25.448 1.00 10.27 C ATOM 1167 OH TYR A 78 19.622 77.324 26.132 1.00 5.54 O ATOM 1169 CE2 TYR A 78 18.975 75.269 24.957 1.00 10.03 C ATOM 1171 CD2 TYR A 78 17.993 74.516 24.210 1.00 10.21 C ATOM 1173 C TYR A 78 17.252 74.731 21.065 1.00 12.05 C ATOM 1174 O TYR A 78 17.672 73.633 20.748 1.00 13.73 O ATOM 1175 N THR A 79 18.004 75.793 20.946 1.00 13.40 N ATOM 1177 CA THR A 79 19.414 75.608 20.674 1.00 14.31 C ATOM 1179 CB THR A 79 19.869 76.190 19.342 1.00 17.06 C ATOM 1181 OG1 THR A 79 19.411 77.531 19.282 1.00 19.07 O ATOM 1183 CG2 THR A 79 19.086 75.686 18.246 1.00 17.91 C ATOM 1187 C THR A 79 20.225 76.294 21.792 1.00 15.43 C ATOM 1188 O THR A 79 21.186 75.669 22.306 1.00 10.46 O ATOM 1189 N SER A 80 19.972 77.563 22.081 1.00 15.52 N ATOM 1191 CA SER A 80 20.640 78.312 23.169 1.00 12.87 C ATOM 1193 CB SER A 80 21.973 78.925 22.719 1.00 15.98 C ATOM 1196 OG SER A 80 21.837 79.840 21.699 1.00 14.14 O ATOM 1198 C SER A 80 19.864 79.500 23.677 1.00 12.72 C ATOM 1199 O SER A 80 18.930 79.946 22.960 1.00 10.08 O ATOM 1200 N GLY A 81 20.375 80.144 24.745 1.00 10.93 N ATOM 1202 CA GLY A 81 19.645 81.253 25.352 1.00 13.73 C ATOM 1205 C GLY A 81 18.495 80.776 26.227 1.00 12.83 C ATOM 1206 O GLY A 81 18.544 79.680 26.738 1.00 13.82 O ATOM 1207 N PHE A 82 17.545 81.650 26.484 1.00 13.00 N ATOM 1209 CA PHE A 82 16.395 81.365 27.314 1.00 13.93 C ATOM 1211 CB PHE A 82 15.515 82.598 27.558 1.00 14.16 C ATOM 1214 CG PHE A 82 16.223 83.611 28.473 1.00 17.70 C ATOM 1215 CD1 PHE A 82 16.312 83.392 29.863 1.00 16.63 C ATOM 1217 CE1 PHE A 82 16.977 84.300 30.671 1.00 18.98 C ATOM 1219 CZ PHE A 82 17.696 85.376 30.113 1.00 18.91 C ATOM 1221 CE2 PHE A 82 17.645 85.629 28.745 1.00 19.29 C ATOM 1223 CD2 PHE A 82 16.931 84.676 27.914 1.00 20.12 C ATOM 1225 C PHE A 82 15.618 80.300 26.564 1.00 14.38 C ATOM 1226 O PHE A 82 15.784 80.111 25.357 1.00 11.55 O ATOM 1227 N ARG A 83 14.815 79.548 27.299 1.00 14.65 N ATOM 1229 CA ARG A 83 13.906 78.568 26.648 1.00 13.87 C ATOM 1231 CB ARG A 83 13.285 77.699 27.778 1.00 14.41 C ATOM 1234 CG ARG A 83 14.287 76.834 28.511 1.00 12.33 C ATOM 1237 CD ARG A 83 13.749 76.304 29.814 1.00 14.68 C ATOM 1240 NE ARG A 83 12.799 75.237 29.520 1.00 15.04 N ATOM 1242 CZ ARG A 83 11.848 74.782 30.335 1.00 18.82 C ATOM 1243 NH1 ARG A 83 11.698 75.249 31.591 1.00 19.87 N ATOM 1246 NH2 ARG A 83 11.000 73.846 29.896 1.00 14.39 N ATOM 1249 C ARG A 83 12.865 79.267 25.827 1.00 12.96 C ATOM 1250 O ARG A 83 12.495 80.387 26.099 1.00 10.42 O ATOM 1251 N ASN A 84 12.289 78.576 24.851 1.00 13.03 N ATOM 1253 CA ASN A 84 11.323 79.134 23.959 1.00 13.01 C ATOM 1255 CB ASN A 84 11.742 78.687 22.560 1.00 12.53 C ATOM 1258 CG ASN A 84 11.750 77.170 22.374 1.00 12.31 C ATOM 1259 OD1 ASN A 84 11.008 76.464 23.075 1.00 9.23 O ATOM 1260 ND2 ASN A 84 12.484 76.678 21.338 1.00 10.53 N ATOM 1263 C ASN A 84 9.966 78.652 24.436 1.00 11.61 C ATOM 1264 O ASN A 84 9.901 78.122 25.523 1.00 10.55 O ATOM 1265 N SER A 85 8.981 78.746 23.580 1.00 11.49 N ATOM 1267 CA SER A 85 7.587 78.334 23.820 1.00 12.14 C ATOM 1269 CB SER A 85 6.672 79.458 23.268 1.00 12.72 C ATOM 1272 OG SER A 85 6.979 80.668 23.991 1.00 15.95 O ATOM 1274 C SER A 85 7.183 76.990 23.312 1.00 10.02 C ATOM 1275 O SER A 85 6.000 76.636 23.266 1.00 12.61 O ATOM 1276 N ASP A 86 8.124 76.267 22.739 1.00 10.38 N ATOM 1278 CA ASP A 86 7.807 75.024 21.992 1.00 12.12 C ATOM 1280 CB ASP A 86 8.722 74.836 20.753 1.00 10.98 C ATOM 1283 CG ASP A 86 8.434 75.862 19.673 1.00 11.98 C ATOM 1284 OD1 ASP A 86 7.319 76.457 19.720 1.00 17.86 O ATOM 1285 OD2 ASP A 86 9.278 76.215 18.820 1.00 15.19 O ATOM 1286 C ASP A 86 8.001 73.810 22.869 1.00 13.31 C ATOM 1287 O ASP A 86 9.112 73.636 23.348 1.00 11.28 O ATOM 1288 N ARG A 87 7.014 72.937 23.016 1.00 12.84 N ATOM 1290 CA ARG A 87 7.255 71.747 23.867 1.00 12.00 C ATOM 1292 CB ARG A 87 6.505 71.952 25.193 1.00 11.15 C ATOM 1295 CG ARG A 87 6.899 73.103 26.000 1.00 12.23 C ATOM 1298 CD ARG A 87 8.318 72.972 26.588 1.00 12.58 C ATOM 1301 NE ARG A 87 8.677 74.154 27.329 1.00 11.57 N ATOM 1303 CZ ARG A 87 9.343 75.184 26.833 1.00 12.24 C ATOM 1304 NH1 ARG A 87 9.763 75.273 25.583 1.00 10.06 N ATOM 1307 NH2 ARG A 87 9.599 76.167 27.669 1.00 14.03 N ATOM 1310 C ARG A 87 6.512 70.600 23.165 1.00 11.13 C ATOM 1311 O ARG A 87 5.416 70.798 22.610 1.00 11.33 O ATOM 1312 N ILE A 88 7.093 69.399 23.231 1.00 13.28 N ATOM 1314 CA ILE A 88 6.374 68.184 22.818 1.00 11.08 C ATOM 1316 CB ILE A 88 7.271 67.247 22.097 1.00 11.24 C ATOM 1318 CG1 ILE A 88 6.492 66.079 21.388 1.00 12.25 C ATOM 1321 CD1 ILE A 88 7.496 65.321 20.368 1.00 12.64 C ATOM 1325 CG2 ILE A 88 8.343 66.729 22.879 1.00 12.59 C ATOM 1329 C ILE A 88 5.833 67.536 24.067 1.00 11.22 C ATOM 1330 O ILE A 88 6.473 67.617 25.116 1.00 10.61 O ATOM 1331 N LEU A 89 4.607 67.018 24.031 1.00 11.36 N ATOM 1333 CA LEU A 89 4.013 66.418 25.233 1.00 7.65 C ATOM 1335 CB LEU A 89 2.676 67.053 25.495 1.00 10.94 C ATOM 1338 CG LEU A 89 2.648 68.432 26.158 1.00 13.79 C ATOM 1340 CD1 LEU A 89 3.199 68.234 27.549 1.00 10.56 C ATOM 1344 CD2 LEU A 89 3.612 69.415 25.636 1.00 15.11 C ATOM 1348 C LEU A 89 3.710 64.952 24.885 1.00 8.75 C ATOM 1349 O LEU A 89 3.147 64.655 23.793 1.00 9.80 O ATOM 1350 N TYR A 90 4.136 64.000 25.721 1.00 9.87 N ATOM 1352 CA TYR A 90 3.927 62.607 25.370 1.00 7.26 C ATOM 1354 CB TYR A 90 5.260 62.014 24.875 1.00 8.09 C ATOM 1357 CG TYR A 90 6.383 62.142 25.902 1.00 7.48 C ATOM 1358 CD1 TYR A 90 7.188 63.276 25.937 1.00 10.00 C ATOM 1360 CE1 TYR A 90 8.334 63.339 26.814 1.00 9.09 C ATOM 1362 CZ TYR A 90 8.740 62.238 27.462 1.00 10.59 C ATOM 1363 OH TYR A 90 9.855 62.410 28.240 1.00 8.93 O ATOM 1365 CE2 TYR A 90 7.993 61.064 27.410 1.00 9.27 C ATOM 1367 CD2 TYR A 90 6.880 61.023 26.507 1.00 8.29 C ATOM 1369 C TYR A 90 3.343 61.906 26.563 1.00 6.89 C ATOM 1370 O TYR A 90 3.700 62.186 27.732 1.00 6.04 O ATOM 1371 N SER A 91 2.368 61.039 26.288 1.00 6.48 N ATOM 1373 CA SER A 91 1.711 60.417 27.421 1.00 7.31 C ATOM 1375 CB SER A 91 0.253 60.198 27.184 1.00 8.94 C ATOM 1378 OG SER A 91 0.034 59.301 26.095 1.00 8.89 O ATOM 1380 C SER A 91 2.252 59.043 27.718 1.00 8.33 C ATOM 1381 O SER A 91 3.011 58.478 26.933 1.00 11.09 O ATOM 1382 N SER A 92 1.899 58.477 28.863 1.00 9.72 N ATOM 1384 CA SER A 92 2.390 57.161 29.299 1.00 9.97 C ATOM 1386 CB SER A 92 2.074 56.942 30.818 1.00 11.10 C ATOM 1389 OG SER A 92 0.656 57.088 30.900 1.00 8.24 O ATOM 1391 C SER A 92 1.817 56.051 28.374 1.00 11.05 C ATOM 1392 O SER A 92 2.383 55.007 28.335 1.00 10.47 O ATOM 1393 N ASP A 93 0.798 56.316 27.543 1.00 9.26 N ATOM 1395 CA ASP A 93 0.286 55.419 26.559 1.00 11.54 C ATOM 1397 CB ASP A 93 −1.228 55.144 26.689 1.00 11.21 C ATOM 1400 CG ASP A 93 −2.095 56.428 26.740 1.00 12.30 C ATOM 1401 OD1 ASP A 93 −3.343 56.275 26.563 1.00 11.48 O ATOM 1402 OD2 ASP A 93 −1.664 57.550 27.003 1.00 11.90 O ATOM 1403 C ASP A 93 0.673 55.946 25.176 1.00 10.07 C ATOM 1404 O ASP A 93 0.104 55.590 24.162 1.00 11.29 O ATOM 1405 N TRP A 94 1.674 56.837 25.143 1.00 11.35 N ATOM 1407 CA TRP A 94 2.197 57.300 23.899 1.00 10.73 C ATOM 1409 CB TRP A 94 3.016 56.177 23.212 1.00 11.67 C ATOM 1412 CG TRP A 94 3.921 55.528 24.202 1.00 11.04 C ATOM 1413 CD1 TRP A 94 3.591 54.410 25.005 1.00 7.89 C ATOM 1415 NE1 TRP A 94 4.628 54.240 25.903 1.00 10.25 N ATOM 1417 CE2 TRP A 94 5.574 55.213 25.732 1.00 7.45 C ATOM 1418 CD2 TRP A 94 5.114 56.099 24.764 1.00 7.97 C ATOM 1419 CE3 TRP A 94 5.941 57.157 24.388 1.00 10.30 C ATOM 1421 CZ3 TRP A 94 7.056 57.427 25.166 1.00 12.75 C ATOM 1423 CH2 TRP A 94 7.458 56.540 26.199 1.00 11.02 C ATOM 1425 CZ2 TRP A 94 6.655 55.477 26.550 1.00 7.10 C ATOM 1427 C TRP A 94 1.474 58.105 22.846 1.00 10.80 C ATOM 1428 O TRP A 94 1.820 57.930 21.666 1.00 12.07 O ATOM 1429 N LEU A 95 0.493 58.902 23.240 1.00 12.80 N ATOM 1431 CA LEU A 95 −0.057 59.966 22.440 1.00 11.32 C ATOM 1433 CB LEU A 95 −1.187 60.616 23.203 1.00 12.25 C ATOM 1436 CG LEU A 95 −2.329 59.624 23.549 1.00 13.74 C ATOM 1438 CD1 LEU A 95 −3.386 60.269 24.382 1.00 15.01 C ATOM 1442 CD2 LEU A 95 −2.918 59.103 22.351 1.00 12.90 C ATOM 1446 C LEU A 95 1.050 61.020 22.438 1.00 12.56 C ATOM 1447 O LEU A 95 1.802 61.072 23.397 1.00 11.37 O ATOM 1448 N ILE A 96 1.130 61.821 21.382 1.00 10.65 N ATOM 1450 CA ILE A 96 2.115 62.885 21.249 1.00 10.60 C ATOM 1452 CB ILE A 96 3.185 62.608 20.202 1.00 9.27 C ATOM 1454 CG1 ILE A 96 3.887 61.268 20.383 1.00 9.17 C ATOM 1457 CD1 ILE A 96 4.677 61.085 21.606 1.00 6.17 C ATOM 1461 CG2 ILE A 96 4.263 63.708 20.223 1.00 10.74 C ATOM 1465 C ILE A 96 1.365 64.113 20.885 1.00 13.34 C ATOM 1466 O ILE A 96 0.580 64.133 19.898 1.00 11.17 O ATOM 1467 N TYR A 97 1.507 65.142 21.698 1.00 10.99 N ATOM 1469 CA TYR A 97 0.948 66.448 21.389 1.00 10.40 C ATOM 1471 CB TYR A 97 −0.071 66.827 22.457 1.00 9.13 C ATOM 1474 CG TYR A 97 −1.476 66.205 22.338 1.00 11.66 C ATOM 1475 CD1 TYR A 97 −1.685 64.842 22.516 1.00 10.97 C ATOM 1477 CE1 TYR A 97 −2.954 64.364 22.408 1.00 10.97 C ATOM 1479 CZ TYR A 97 −4.022 65.180 22.159 1.00 9.89 C ATOM 1480 OH TYR A 97 −5.198 64.549 22.083 1.00 12.29 O ATOM 1482 CE2 TYR A 97 −3.858 66.528 21.939 1.00 8.64 C ATOM 1484 CD2 TYR A 97 −2.565 66.987 21.999 1.00 9.70 C ATOM 1486 C TYR A 97 2.111 67.484 21.432 1.00 9.53 C ATOM 1487 O TYR A 97 3.248 67.144 21.660 1.00 14.36 O ATOM 1488 N LYS A 98 1.871 68.645 20.840 1.00 11.28 N ATOM 1490 CA LYS A 98 2.723 69.809 20.826 1.00 8.69 C ATOM 1492 CB LYS A 98 3.322 70.137 19.432 1.00 10.99 C ATOM 1495 CG LYS A 98 2.287 70.453 18.429 1.00 11.17 C ATOM 1498 CD LYS A 98 2.737 70.559 17.046 1.00 12.80 C ATOM 1501 CE LYS A 98 3.596 71.735 16.780 1.00 14.37 C ATOM 1504 NZ LYS A 98 3.792 72.136 15.359 1.00 11.80 N ATOM 1508 C LYS A 98 1.962 71.062 21.287 1.00 9.67 C ATOM 1509 O LYS A 98 0.705 71.178 21.200 1.00 10.69 O ATOM 1510 N THR A 99 2.802 71.949 21.791 1.00 11.10 N ATOM 1512 CA THR A 99 2.478 73.322 22.083 1.00 11.12 C ATOM 1514 CB THR A 99 2.279 73.630 23.530 1.00 11.10 C ATOM 1516 OG1 THR A 99 1.848 75.009 23.663 1.00 12.60 O ATOM 1518 CG2 THR A 99 3.664 73.409 24.231 1.00 14.35 C ATOM 1522 C THR A 99 3.531 74.204 21.466 1.00 9.24 C ATOM 1523 O THR A 99 4.699 73.886 21.462 1.00 12.21 O ATOM 1524 N THR A 100 3.142 75.275 20.791 1.00 10.21 N ATOM 1526 CA THR A 100 4.085 76.343 20.391 1.00 7.68 C ATOM 1528 CB THR A 100 4.035 76.517 18.912 1.00 8.62 C ATOM 1530 OG1 THR A 100 2.746 76.881 18.484 1.00 6.34 O ATOM 1532 CG2 THR A 100 4.131 75.258 18.267 1.00 10.31 C ATOM 1536 C THR A 100 3.816 77.679 21.115 1.00 10.03 C ATOM 1537 O THR A 100 4.344 78.734 20.713 1.00 8.97 O ATOM 1538 N ASP A 101 2.888 77.679 22.099 1.00 12.17 N ATOM 1540 CA ASP A 101 2.480 78.926 22.827 1.00 11.94 C ATOM 1542 CB ASP A 101 1.111 79.438 22.366 1.00 12.53 C ATOM 1545 CG ASP A 101 0.017 78.428 22.586 1.00 12.67 C ATOM 1546 OD1 ASP A 101 0.248 77.401 23.288 1.00 8.36 O ATOM 1547 OD2 ASP A 101 −1.154 78.592 22.146 1.00 16.13 O ATOM 1548 C ASP A 101 2.637 78.764 24.353 1.00 12.93 C ATOM 1549 O ASP A 101 1.823 79.275 25.139 1.00 13.24 O ATOM 1550 N ALA A 102 3.625 77.957 24.771 1.00 11.21 N ATOM 1552 CA ALA A 102 3.961 77.793 26.197 1.00 13.13 C ATOM 1554 CB ALA A 102 4.537 79.124 26.793 1.00 15.01 C ATOM 1558 C ALA A 102 2.768 77.206 26.945 1.00 12.35 C ATOM 1559 O ALA A 102 2.301 77.790 27.907 1.00 8.75 O ATOM 1560 N TYR A 103 2.246 76.082 26.425 1.00 8.76 N ATOM 1562 CA TYR A 103 1.140 75.333 27.104 1.00 12.19 C ATOM 1564 CB TYR A 103 1.476 75.002 28.562 1.00 15.30 C ATOM 1567 CG TYR A 103 2.919 74.643 28.825 1.00 14.89 C ATOM 1568 CD1 TYR A 103 3.379 73.369 28.624 1.00 16.53 C ATOM 1570 CE1 TYR A 103 4.731 73.030 28.872 1.00 15.54 C ATOM 1572 CZ TYR A 103 5.578 73.972 29.301 1.00 14.65 C ATOM 1573 OH TYR A 103 6.856 73.644 29.477 1.00 10.93 O ATOM 1575 CE2 TYR A 103 5.166 75.245 29.529 1.00 17.37 C ATOM 1577 CD2 TYR A 103 3.819 75.586 29.288 1.00 17.19 C ATOM 1579 C TYR A 103 −0.261 75.975 27.097 1.00 14.30 C ATOM 1580 O TYR A 103 −1.242 75.482 27.722 1.00 11.25 O ATOM 1581 N GLN A 104 −0.401 77.056 26.344 1.00 15.15 N ATOM 1583 CA GLN A 104 −1.727 77.638 26.300 1.00 16.77 C ATOM 1585 CB GLN A 104 −1.639 79.064 25.757 1.00 20.34 C ATOM 1588 CG GLN A 104 −0.796 80.074 26.634 1.00 25.22 C ATOM 1591 CD GLN A 104 −0.824 81.541 26.069 1.00 28.73 C ATOM 1592 OE1 GLN A 104 −1.846 82.229 26.191 1.00 29.45 O ATOM 1593 NE2 GLN A 104 0.280 81.975 25.393 1.00 30.51 N ATOM 1596 C GLN A 104 −2.675 76.767 25.445 1.00 14.55 C ATOM 1597 O GLN A 104 −3.878 76.706 25.696 1.00 15.19 O ATOM 1598 N THR A 105 −2.185 76.259 24.328 1.00 12.62 N ATOM 1600 CA THR A 105 −3.004 75.402 23.422 1.00 11.08 C ATOM 1602 CB THR A 105 −3.509 76.107 22.187 1.00 10.97 C ATOM 1604 OG1 THR A 105 −2.423 76.595 21.389 1.00 11.27 O ATOM 1606 CG2 THR A 105 −4.288 77.422 22.549 1.00 11.24 C ATOM 1610 C THR A 105 −2.092 74.272 22.929 1.00 12.45 C ATOM 1611 O THR A 105 −0.851 74.392 22.878 1.00 11.77 O ATOM 1612 N PHE A 106 −2.745 73.162 22.581 1.00 12.46 N ATOM 1614 CA PHE A 106 −2.052 71.958 22.188 1.00 13.94 C ATOM 1616 CB PHE A 106 −2.241 70.958 23.300 1.00 15.04 C ATOM 1619 CG PHE A 106 −1.644 71.393 24.580 1.00 17.01 C ATOM 1620 CD1 PHE A 106 −0.348 71.055 24.876 1.00 17.25 C ATOM 1622 CE1 PHE A 106 0.203 71.431 26.083 1.00 19.41 C ATOM 1624 CZ PHE A 106 −0.596 72.104 27.017 1.00 18.42 C ATOM 1626 CE2 PHE A 106 −1.924 72.363 26.748 1.00 17.27 C ATOM 1628 CD2 PHE A 106 −2.426 72.041 25.525 1.00 18.01 C ATOM 1630 C PHE A 106 −2.624 71.412 20.915 1.00 12.39 C ATOM 1631 O PHE A 106 −3.806 71.598 20.631 1.00 10.59 O ATOM 1632 N THR A 107 −1.790 70.681 20.182 1.00 8.23 N ATOM 1634 CA THR A 107 −2.252 70.110 18.939 1.00 8.80 C ATOM 1636 CB THR A 107 −1.544 70.769 17.737 1.00 8.29 C ATOM 1638 OG1 THR A 107 −1.941 72.116 17.663 1.00 6.57 O ATOM 1640 CG2 THR A 107 −1.888 70.188 16.328 1.00 9.11 C ATOM 1644 C THR A 107 −1.722 68.685 18.979 1.00 9.08 C ATOM 1645 O THR A 107 −0.526 68.448 19.214 1.00 8.70 O ATOM 1646 N LYS A 108 −2.644 67.752 18.641 1.00 7.72 N ATOM 1648 CA LYS A 108 −2.313 66.334 18.620 1.00 10.16 C ATOM 1650 CB LYS A 108 −3.567 65.422 18.581 1.00 9.16 C ATOM 1653 CG LYS A 108 −3.278 63.953 18.663 1.00 7.26 C ATOM 1656 CD LYS A 108 −4.645 63.226 18.826 1.00 8.61 C ATOM 1659 CE LYS A 108 −4.416 61.782 18.664 1.00 11.16 C ATOM 1662 NZ LYS A 108 −5.542 60.906 19.154 1.00 5.13 N ATOM 1666 C LYS A 108 −1.515 66.072 17.398 1.00 8.51 C ATOM 1667 O LYS A 108 −1.983 66.354 16.308 1.00 10.06 O ATOM 1668 N ILE A 109 −0.374 65.450 17.573 1.00 7.29 N ATOM 1670 CA ILE A 109 0.458 65.093 16.441 1.00 10.46 C ATOM 1672 CB ILE A 109 1.838 65.833 16.410 1.00 8.44 C ATOM 1674 CG1 ILE A 109 2.710 65.557 17.634 1.00 10.16 C ATOM 1677 CD1 ILE A 109 4.081 66.272 17.332 1.00 7.91 C ATOM 1681 CG2 ILE A 109 1.599 67.357 16.199 1.00 12.74 C ATOM 1685 C ILE A 109 0.773 63.629 16.184 1.00 12.02 C ATOM 1686 O ILE A 109 1.414 63.338 15.178 1.00 8.52 O ATOM 1687 N ARG A 110 0.572 62.794 17.193 1.00 13.26 N ATOM 1689 CA ARG A 110 0.591 61.359 16.992 1.00 13.26 C ATOM 1691 CB ARG A 110 1.890 60.663 17.316 1.00 11.90 C ATOM 1694 CG ARG A 110 3.052 61.153 16.489 1.00 11.81 C ATOM 1697 CD ARG A 110 3.152 60.822 15.016 1.00 9.75 C ATOM 1700 NE ARG A 110 4.549 61.145 14.559 1.00 5.77 N ATOM 1702 CZ ARG A 110 4.863 62.377 14.133 1.00 5.92 C ATOM 1703 NH1 ARG A 110 3.983 63.411 14.119 1.00 8.31 N ATOM 1706 NH2 ARG A 110 6.026 62.568 13.645 1.00 5.94 N ATOM 1709 C ARG A 110 −0.519 60.667 17.721 1.00 11.42 C ATOM 1710 O ARG A 110 −0.696 61.007 18.840 1.00 8.70 O ATOM 1711 N SER A 111 −1.209 59.704 17.091 1.00 14.26 N ATOM 1713 CA SER A 111 −1.958 58.641 17.754 1.00 17.02 C ATOM 1715 CB SER A 111 −2.724 57.712 16.723 1.00 20.16 C ATOM 1718 OG SER A 111 −3.743 58.582 16.265 1.00 21.74 O ATOM 1720 C SER A 111 −0.919 57.829 18.544 1.00 14.70 C ATOM 1721 O SER A 111 0.273 57.836 18.212 1.00 16.86 O ATOM 1722 N SER A 112 −1.445 57.078 19.469 1.00 15.33 N ATOM 1724 CA SER A 112 −0.660 56.086 20.204 1.00 16.21 C ATOM 1726 CB SER A 112 −1.475 55.242 21.153 1.00 16.36 C ATOM 1729 OG SER A 112 −0.683 54.311 21.872 1.00 15.39 O ATOM 1731 C SER A 112 −0.040 55.123 19.269 1.00 16.70 C ATOM 1732 O SER A 112 −0.684 54.627 18.315 1.00 20.34 O ATOM 1733 N SER A 113 1.299 54.994 19.419 1.00 17.37 N ATOM 1735 CA SER A 113 2.086 53.975 18.751 1.00 15.72 C ATOM 1737 CB SER A 113 3.506 54.569 18.467 1.00 16.57 C ATOM 1740 OG SER A 113 3.954 54.927 19.739 1.00 16.43 O ATOM 1742 C SER A 113 2.165 52.631 19.489 1.00 14.13 C ATOM 1743 O SER A 113 2.698 51.694 18.980 1.00 15.35 O ATOM 1744 N MET A 114 1.461 52.409 20.599 1.00 13.09 N ATOM 1746 CA MET A 114 1.406 51.112 21.222 1.00 12.38 C ATOM 1748 CB MET A 114 0.459 51.169 22.415 1.00 15.07 C ATOM 1751 CG MET A 114 1.178 52.022 23.536 1.00 19.87 C ATOM 1754 SD MET A 114 0.264 52.200 25.023 1.00 21.07 S ATOM 1755 CE MET A 114 0.298 50.494 25.805 1.00 23.70 C ATOM 1759 C MET A 114 0.886 50.017 20.327 1.00 15.06 C ATOM 1760 O MET A 114 −0.020 50.332 19.607 1.00 15.11 O ATOM 1761 N GLY A 115 1.654 48.922 20.220 1.00 9.36 N ATOM 1763 CA GLY A 115 1.268 47.835 19.378 1.00 10.49 C ATOM 1766 C GLY A 115 1.949 47.868 18.035 1.00 10.29 C ATOM 1767 O GLY A 115 1.864 46.856 17.297 1.00 10.82 O ATOM 1768 N VAL A 116 2.505 49.022 17.688 1.00 7.02 N ATOM 1770 CA VAL A 116 3.178 49.172 16.391 1.00 9.43 C ATOM 1772 CB VAL A 116 3.052 50.614 15.783 1.00 10.47 C ATOM 1774 CG1 VAL A 116 3.941 50.829 14.510 1.00 10.78 C ATOM 1778 CG2 VAL A 116 1.642 51.035 15.568 1.00 10.20 C ATOM 1782 C VAL A 116 4.655 48.797 16.548 1.00 8.38 C ATOM 1783 O VAL A 116 5.294 49.372 17.399 1.00 10.29 O ATOM 1784 N CYS A 117 5.229 47.931 15.688 1.00 5.54 N ATOM 1786 CA CYS A 117 6.624 47.576 15.832 1.00 6.25 C ATOM 1788 CB CYS A 117 6.677 46.052 15.502 1.00 5.28 C ATOM 1791 SG CYS A 117 8.354 45.441 15.452 1.00 8.44 S ATOM 1792 C CYS A 117 7.411 48.304 14.769 1.00 9.64 C ATOM 1793 O CYS A 117 7.075 48.159 13.586 1.00 10.64 O ATOM 1794 N PRO A 118 8.347 49.144 15.166 1.00 9.31 N ATOM 1795 CA PRO A 118 9.143 49.923 14.228 1.00 10.70 C ATOM 1797 CB PRO A 118 10.164 50.594 15.173 1.00 8.88 C ATOM 1800 CG PRO A 118 9.315 50.725 16.437 1.00 10.00 C ATOM 1803 CD PRO A 118 8.747 49.349 16.572 1.00 6.82 C ATOM 1806 C PRO A 118 9.838 48.934 13.269 1.00 10.27 C ATOM 1807 O PRO A 118 10.321 47.864 13.627 1.00 10.68 O ATOM 1808 N LYS A 119 10.089 49.409 12.064 1.00 12.58 N ATOM 1810 CA LYS A 119 10.829 48.643 11.060 1.00 12.76 C ATOM 1812 CB LYS A 119 10.568 49.228 9.653 1.00 15.68 C ATOM 1815 CG LYS A 119 11.272 48.555 8.463 1.00 16.64 C ATOM 1818 CD LYS A 119 11.111 46.997 8.542 1.00 18.87 C ATOM 1821 CE LYS A 119 9.842 46.280 7.993 1.00 18.06 C ATOM 1824 NZ LYS A 119 9.763 44.824 8.359 1.00 17.21 N ATOM 1828 C LYS A 119 12.298 48.788 11.359 1.00 14.02 C ATOM 1829 O LYS A 119 13.043 49.620 10.835 1.00 15.49 O ATOM 1830 N ILE A 120 12.769 47.964 12.290 1.00 13.51 N ATOM 1832 CA ILE A 120 14.178 48.094 12.694 1.00 9.14 C ATOM 1834 CB ILE A 120 14.248 48.834 14.049 1.00 11.23 C ATOM 1836 CG1 ILE A 120 14.036 50.319 13.716 1.00 13.36 C ATOM 1839 CD1 ILE A 120 14.146 51.261 14.818 1.00 11.19 C ATOM 1843 CG2 ILE A 120 15.702 48.607 14.704 1.00 11.91 C ATOM 1847 C ILE A 120 14.643 46.647 12.985 1.00 9.77 C ATOM 1848 O ILE A 120 13.918 45.854 13.625 1.00 9.24 O ATOM 1849 N LEU A 121 15.794 46.275 12.450 1.00 9.71 N ATOM 1851 CA LEU A 121 16.298 44.919 12.478 1.00 11.68 C ATOM 1853 CB LEU A 121 17.380 44.869 11.407 1.00 15.05 C ATOM 1856 CG LEU A 121 17.667 43.536 10.800 1.00 18.59 C ATOM 1858 CD1 LEU A 121 16.393 42.688 10.319 1.00 20.06 C ATOM 1862 CD2 LEU A 121 18.691 43.933 9.706 1.00 21.03 C ATOM 1866 C LEU A 121 16.944 44.749 13.830 1.00 13.87 C ATOM 1867 O LEU A 121 17.821 45.527 14.222 1.00 12.91 O ATOM 1868 N LYS A 122 16.425 43.790 14.582 1.00 13.07 N ATOM 1870 CA LYS A 122 16.842 43.595 15.962 1.00 13.86 C ATOM 1872 CB LYS A 122 15.956 44.394 16.954 1.00 14.08 C ATOM 1875 CG LYS A 122 16.400 44.389 18.454 1.00 19.57 C ATOM 1878 CD LYS A 122 17.852 44.689 18.809 1.00 21.48 C ATOM 1881 CE LYS A 122 18.343 44.220 20.262 1.00 24.39 C ATOM 1884 NZ LYS A 122 19.234 42.945 20.283 1.00 22.59 N ATOM 1888 C LYS A 122 16.860 42.115 16.279 1.00 9.69 C ATOM 1889 O LYS A 122 15.856 41.522 16.259 1.00 10.32 O ATOM 1890 N LYS A 123 17.944 41.530 16.778 1.00 7.24 N ATOM 1892 CA LYS A 123 17.910 40.161 17.158 1.00 7.74 C ATOM 1894 CB LYS A 123 19.343 39.669 17.314 1.00 8.85 C ATOM 1897 CG LYS A 123 20.025 39.680 15.990 1.00 9.77 C ATOM 1900 CD LYS A 123 21.433 39.034 16.157 1.00 13.94 C ATOM 1903 CE LYS A 123 21.966 38.563 14.829 1.00 17.19 C ATOM 1906 NZ LYS A 123 22.187 39.772 13.984 1.00 16.89 N ATOM 1910 C LYS A 123 17.318 40.061 18.540 1.00 8.95 C ATOM 1911 O LYS A 123 17.459 40.947 19.394 1.00 7.12 O ATOM 1912 N CYS A 124 16.709 38.917 18.791 1.00 10.81 N ATOM 1914 CA CYS A 124 16.102 38.662 20.074 1.00 10.25 C ATOM 1916 CB CYS A 124 14.679 39.198 20.074 1.00 9.54 C ATOM 1919 SG CYS A 124 13.811 38.420 18.689 1.00 8.73 S ATOM 1920 C CYS A 124 16.097 37.162 20.431 1.00 10.85 C ATOM 1921 O CYS A 124 16.179 36.263 19.594 1.00 9.26 O ATOM 1922 N ARG A 125 15.845 36.927 21.706 1.00 12.03 N ATOM 1924 CA ARG A 125 15.494 35.602 22.261 1.00 14.18 C ATOM 1926 CB ARG A 125 16.547 35.142 23.312 1.00 18.03 C ATOM 1929 CG ARG A 125 17.839 34.572 22.673 1.00 23.34 C ATOM 1932 CD ARG A 125 19.191 34.637 23.511 1.00 28.34 C ATOM 1935 NE ARG A 125 20.180 33.556 23.222 1.00 31.12 N ATOM 1937 CZ ARG A 125 19.928 32.262 23.506 1.00 34.85 C ATOM 1938 NH1 ARG A 125 18.765 31.934 24.100 1.00 37.06 N ATOM 1941 NH2 ARG A 125 20.787 31.281 23.235 1.00 35.19 N ATOM 1944 C ARG A 125 14.066 35.670 22.883 1.00 12.22 C ATOM 1945 O ARG A 125 13.409 34.674 22.927 1.00 7.31 O ATOM 1946 N ARG A 126 13.522 36.830 23.240 1.00 12.86 N ATOM 1948 CA ARG A 126 12.234 36.928 23.840 1.00 14.85 C ATOM 1950 CB ARG A 126 12.297 36.637 25.331 1.00 17.94 C ATOM 1953 CG ARG A 126 13.257 37.535 26.061 1.00 19.22 C ATOM 1956 CD ARG A 126 13.090 37.583 27.592 1.00 23.45 C ATOM 1959 NE ARG A 126 11.702 37.396 28.055 1.00 26.65 N ATOM 1961 CZ ARG A 126 11.341 36.720 29.159 1.00 29.80 C ATOM 1962 NH1 ARG A 126 12.190 36.104 29.976 1.00 29.40 N ATOM 1965 NH2 ARG A 126 10.054 36.585 29.456 1.00 31.54 N ATOM 1968 C ARG A 126 11.723 38.301 23.507 1.00 13.43 C ATOM 1969 O ARG A 126 12.499 39.161 23.074 1.00 11.02 O ATOM 1970 N ASP A 127 10.432 38.511 23.751 1.00 8.99 N ATOM 1972 CA ASP A 127 9.815 39.799 23.369 1.00 9.96 C ATOM 1974 CB ASP A 127 8.301 39.815 23.730 1.00 9.55 C ATOM 1977 CG ASP A 127 7.539 38.785 22.893 1.00 10.42 C ATOM 1978 OD1 ASP A 127 6.398 38.465 23.319 1.00 8.22 O ATOM 1979 OD2 ASP A 127 8.157 38.107 21.987 1.00 9.63 O ATOM 1980 C ASP A 127 10.507 41.011 23.967 1.00 6.95 C ATOM 1981 O ASP A 127 10.617 42.050 23.351 1.00 4.94 O ATOM 1982 N SER A 128 10.916 40.905 25.209 1.00 9.26 N ATOM 1984 CA SER A 128 11.467 42.058 25.882 1.00 10.08 C ATOM 1986 CB SER A 128 11.599 41.822 27.397 1.00 12.52 C ATOM 1989 OG SER A 128 12.320 40.648 27.732 1.00 11.88 O ATOM 1991 C SER A 128 12.779 42.492 25.315 1.00 9.99 C ATOM 1992 O SER A 128 13.299 43.506 25.763 1.00 9.39 O ATOM 1993 N ASP A 129 13.332 41.713 24.389 1.00 9.66 N ATOM 1995 CA ASP A 129 14.612 42.072 23.757 1.00 8.80 C ATOM 1997 CB ASP A 129 15.291 40.884 23.196 1.00 7.88 C ATOM 2000 CG ASP A 129 15.869 39.997 24.229 1.00 9.83 C ATOM 2001 OD1 ASP A 129 16.317 40.424 25.331 1.00 12.63 O ATOM 2002 OD2 ASP A 129 16.023 38.817 23.895 1.00 7.97 O ATOM 2003 C ASP A 129 14.247 42.999 22.580 1.00 10.66 C ATOM 2004 O ASP A 129 15.044 43.734 22.035 1.00 9.95 O ATOM 2005 N CYS A 130 12.966 43.125 22.304 1.00 9.69 N ATOM 2007 CA CYS A 130 12.596 43.961 21.202 1.00 10.40 C ATOM 2009 CB CYS A 130 11.546 43.241 20.368 1.00 10.66 C ATOM 2012 SG CYS A 130 11.959 41.602 19.702 1.00 10.81 S ATOM 2013 C CYS A 130 11.939 45.265 21.648 1.00 9.65 C ATOM 2014 O CYS A 130 11.417 45.435 22.752 1.00 12.43 O ATOM 2015 N LEU A 131 11.768 46.141 20.680 1.00 8.80 N ATOM 2017 CA LEU A 131 11.162 47.419 20.905 1.00 9.00 C ATOM 2019 CB LEU A 131 11.232 48.250 19.620 1.00 9.08 C ATOM 2022 CG LEU A 131 12.641 48.674 19.336 1.00 7.59 C ATOM 2024 CD1 LEU A 131 12.884 49.341 18.066 1.00 8.36 C ATOM 2028 CD2 LEU A 131 13.314 49.481 20.457 1.00 9.67 C ATOM 2032 C LEU A 131 9.692 47.224 21.237 1.00 8.20 C ATOM 2033 O LEU A 131 9.069 46.252 20.882 1.00 9.52 O ATOM 2034 N ALA A 132 9.123 48.267 21.809 1.00 5.72 N ATOM 2036 CA ALA A 132 7.712 48.167 22.036 1.00 6.14 C ATOM 2038 CB ALA A 132 7.272 49.392 22.713 1.00 4.65 C ATOM 2042 C ALA A 132 6.907 47.955 20.754 1.00 5.20 C ATOM 2043 O ALA A 132 7.109 48.660 19.731 1.00 7.05 O ATOM 2044 N GLY A 133 5.836 47.158 20.890 1.00 7.26 N ATOM 2046 CA GLY A 133 4.986 46.833 19.773 1.00 6.59 C ATOM 2049 C GLY A 133 5.551 45.630 18.983 1.00 7.38 C ATOM 2050 O GLY A 133 4.854 45.143 18.095 1.00 7.56 O ATOM 2051 N CYS A 134 6.715 45.096 19.316 1.00 6.87 N ATOM 2053 CA CYS A 134 7.395 44.072 18.598 1.00 6.03 C ATOM 2055 CB CYS A 134 8.849 44.449 18.295 1.00 7.35 C ATOM 2058 SG CYS A 134 9.007 45.911 17.270 1.00 8.44 S ATOM 2059 C CYS A 134 7.412 42.795 19.456 1.00 7.86 C ATOM 2060 O CYS A 134 7.613 42.827 20.697 1.00 7.10 O ATOM 2061 N VAL A 135 7.563 41.673 18.743 1.00 8.68 N ATOM 2063 CA VAL A 135 7.636 40.395 19.396 1.00 11.71 C ATOM 2065 CB VAL A 135 6.326 39.568 19.330 1.00 12.59 C ATOM 2067 CG1 VAL A 135 5.239 40.261 20.132 1.00 12.03 C ATOM 2071 CG2 VAL A 135 5.942 39.183 17.931 1.00 11.54 C ATOM 2075 C VAL A 135 8.754 39.610 18.731 1.00 12.89 C ATOM 2076 O VAL A 135 9.193 39.949 17.638 1.00 15.44 O ATOM 2077 N CYS A 136 9.347 38.690 19.514 1.00 12.32 N ATOM 2079 CA CYS A 136 10.425 37.864 19.038 1.00 9.14 C ATOM 2081 CB CYS A 136 11.351 37.414 20.162 1.00 9.24 C ATOM 2084 SG CYS A 136 12.853 36.730 19.489 1.00 9.57 S ATOM 2085 C CYS A 136 9.877 36.721 18.191 1.00 9.81 C ATOM 2086 O CYS A 136 8.999 35.948 18.560 1.00 10.22 O ATOM 2087 N GLY A 137 10.384 36.666 16.958 1.00 11.30 N ATOM 2089 CA GLY A 137 9.914 35.695 15.948 1.00 11.53 C ATOM 2092 C GLY A 137 10.704 34.425 15.986 1.00 10.87 C ATOM 2093 O GLY A 137 11.594 34.309 16.806 1.00 10.53 O ATOM 2094 N PRO A 138 10.362 33.432 15.175 1.00 10.27 N ATOM 2095 CA PRO A 138 11.060 32.150 15.353 1.00 10.62 C ATOM 2097 CB PRO A 138 10.114 31.148 14.663 1.00 11.02 C ATOM 2100 CG PRO A 138 9.523 32.010 13.541 1.00 9.49 C ATOM 2103 CD PRO A 138 9.185 33.314 14.275 1.00 9.58 C ATOM 2106 C PRO A 138 12.461 32.121 14.789 1.00 10.49 C ATOM 2107 O PRO A 138 13.013 31.031 14.849 1.00 7.78 O ATOM 2108 N ASN A 139 12.955 33.188 14.131 1.00 10.69 N ATOM 2110 CA ASN A 139 14.279 33.178 13.637 1.00 8.04 C ATOM 2112 CB ASN A 139 14.355 33.625 12.201 1.00 8.85 C ATOM 2115 CG ASN A 139 14.099 32.486 11.250 1.00 11.43 C ATOM 2116 OD1 ASN A 139 13.582 31.405 11.673 1.00 12.02 O ATOM 2117 ND2 ASN A 139 14.506 32.691 9.974 1.00 8.01 N ATOM 2120 C ASN A 139 15.231 34.047 14.428 1.00 10.21 C ATOM 2121 O ASN A 139 16.365 34.344 14.006 1.00 11.74 O ATOM 2122 N GLY A 140 14.815 34.372 15.607 1.00 9.67 N ATOM 2124 CA GLY A 140 15.589 35.221 16.463 1.00 10.04 C ATOM 2127 C GLY A 140 15.547 36.669 15.998 1.00 10.48 C ATOM 2128 O GLY A 140 16.426 37.390 16.407 1.00 9.32 O ATOM 2129 N PHE A 141 14.528 37.111 15.260 1.00 10.21 N ATOM 2131 CA PHE A 141 14.453 38.536 14.939 1.00 10.80 C ATOM 2133 CB PHE A 141 14.723 38.821 13.455 1.00 10.24 C ATOM 2136 CG PHE A 141 16.147 38.733 13.087 1.00 10.69 C ATOM 2137 CD1 PHE A 141 16.931 39.862 13.114 1.00 11.83 C ATOM 2139 CE1 PHE A 141 18.217 39.837 12.812 1.00 10.05 C ATOM 2141 CZ PHE A 141 18.775 38.651 12.386 1.00 7.45 C ATOM 2143 CE2 PHE A 141 18.049 37.509 12.410 1.00 6.60 C ATOM 2145 CD2 PHE A 141 16.752 37.523 12.759 1.00 9.67 C ATOM 2147 C PHE A 141 13.132 39.101 15.382 1.00 11.58 C ATOM 2148 O PHE A 141 12.080 38.398 15.451 1.00 14.86 O ATOM 2149 N CYS A 142 13.184 40.385 15.742 1.00 10.60 N ATOM 2151 CA CYS A 142 11.979 41.109 16.147 1.00 10.20 C ATOM 2153 CB CYS A 142 12.321 42.364 16.941 1.00 10.04 C ATOM 2156 SG CYS A 142 13.395 42.150 18.391 1.00 8.87 S ATOM 2157 C CYS A 142 11.030 41.473 15.017 1.00 9.93 C ATOM 2158 O CYS A 142 11.490 41.746 13.959 1.00 12.30 O ATOM 2159 N GLY A 143 9.690 41.467 15.215 1.00 6.38 N ATOM 2161 CA GLY A 143 8.764 41.871 14.194 1.00 8.97 C ATOM 2164 C GLY A 143 7.380 42.086 14.827 1.00 9.03 C ATOM 2165 O GLY A 143 7.277 42.013 16.034 1.00 4.04 O ATOM 2166 N SER A 144 6.399 42.455 14.038 1.00 6.51 N ATOM 2168 CA SER A 144 5.072 42.806 14.519 1.00 8.21 C ATOM 2170 CB SER A 144 4.211 43.276 13.280 1.00 6.85 C ATOM 2173 OG SER A 144 4.868 44.313 12.534 1.00 8.90 O ATOM 2175 C SER A 144 4.545 41.514 15.054 1.00 11.77 C ATOM 2176 O SER A 144 4.836 40.367 14.657 1.00 13.97 O ATOM 2177 OXT SER A 144 3.644 41.553 15.871 1.00 17.62 O ATOM 2178 N GLN B 2 −7.652 18.523 35.984 1.00 47.13 N ATOM 2180 CA GLN B 2 −6.606 19.347 36.701 1.00 46.13 C ATOM 2182 CB GLN B 2 −6.839 19.145 38.193 1.00 48.68 C ATOM 2185 CG GLN B 2 −8.350 19.427 38.618 1.00 51.28 C ATOM 2188 CD GLN B 2 −9.116 18.300 39.436 1.00 53.84 C ATOM 2189 OE1 GLN B 2 −9.170 18.307 40.693 1.00 55.09 O ATOM 2190 NE2 GLN B 2 −9.777 17.382 38.706 1.00 54.79 N ATOM 2193 C GLN B 2 −5.133 19.105 36.229 1.00 42.32 C ATOM 2194 O GLN B 2 −4.350 20.059 36.029 1.00 40.81 O ATOM 2197 N VAL B 3 −4.756 17.840 35.983 1.00 36.38 N ATOM 2199 CA VAL B 3 −3.433 17.513 35.386 1.00 29.41 C ATOM 2201 CB VAL B 3 −3.025 16.089 35.713 1.00 30.90 C ATOM 2203 CG1 VAL B 3 −2.293 15.320 34.648 1.00 29.79 C ATOM 2207 CG2 VAL B 3 −2.305 16.044 37.089 1.00 32.67 C ATOM 2211 C VAL B 3 −3.532 17.692 33.876 1.00 22.75 C ATOM 2212 O VAL B 3 −4.500 17.320 33.231 1.00 20.09 O ATOM 2213 N ILE B 4 −2.494 18.302 33.309 1.00 17.63 N ATOM 2215 CA ILE B 4 −2.374 18.510 31.862 1.00 14.01 C ATOM 2217 CB ILE B 4 −2.148 19.960 31.544 1.00 13.99 C ATOM 2219 CG1 ILE B 4 −3.252 20.848 32.157 1.00 15.54 C ATOM 2222 CD1 ILE B 4 −3.042 22.418 31.823 1.00 14.33 C ATOM 2226 CG2 ILE B 4 −2.030 20.158 30.050 1.00 12.10 C ATOM 2230 C ILE B 4 −1.247 17.645 31.388 1.00 11.29 C ATOM 2231 O ILE B 4 −0.065 17.851 31.711 1.00 12.25 O ATOM 2232 N ASN B 5 −1.642 16.591 30.683 1.00 10.98 N ATOM 2234 CA ASN B 5 −0.749 15.581 30.162 1.00 7.79 C ATOM 2236 CB ASN B 5 −0.427 14.539 31.159 1.00 7.24 C ATOM 2239 CG ASN B 5 −1.512 13.458 31.336 1.00 9.99 C ATOM 2240 OD1 ASN B 5 −1.283 12.447 32.020 1.00 14.44 O ATOM 2241 ND2 ASN B 5 −2.679 13.706 30.877 1.00 5.64 N ATOM 2244 C ASN B 5 −1.121 14.874 28.892 1.00 9.61 C ATOM 2245 O ASN B 5 −0.656 13.757 28.698 1.00 11.34 O ATOM 2246 N THR B 6 −1.998 15.447 28.088 1.00 9.86 N ATOM 2248 CA THR B 6 −2.366 14.887 26.817 1.00 8.91 C ATOM 2250 CB THR B 6 −3.818 15.075 26.457 1.00 6.42 C ATOM 2252 OG1 THR B 6 −4.741 14.395 27.429 1.00 14.51 O ATOM 2254 CG2 THR B 6 −4.110 16.432 26.411 1.00 2.00 C ATOM 2258 C THR B 6 −1.455 15.671 25.807 1.00 10.95 C ATOM 2259 O THR B 6 −0.921 16.748 26.054 1.00 9.64 O ATOM 2260 N PHE B 7 −1.292 15.106 24.618 1.00 10.00 N ATOM 2262 CA PHE B 7 −0.470 15.699 23.620 1.00 8.08 C ATOM 2264 CB PHE B 7 −0.499 14.884 22.342 1.00 8.24 C ATOM 2267 CG PHE B 7 0.343 13.605 22.376 1.00 9.14 C ATOM 2268 CD1 PHE B 7 1.714 13.666 22.366 1.00 10.72 C ATOM 2270 CE1 PHE B 7 2.478 12.542 22.402 1.00 12.63 C ATOM 2272 CZ PHE B 7 1.929 11.326 22.385 1.00 11.51 C ATOM 2274 CE2 PHE B 7 0.539 11.202 22.442 1.00 11.44 C ATOM 2276 CD2 PHE B 7 −0.263 12.356 22.468 1.00 10.83 C ATOM 2278 C PHE B 7 −1.049 17.081 23.251 1.00 8.06 C ATOM 2279 O PHE B 7 −0.322 18.035 23.253 1.00 7.93 O ATOM 2280 N ASP B 8 −2.321 17.256 22.965 1.00 6.86 N ATOM 2282 CA ASP B 8 −2.830 18.561 22.606 1.00 10.26 C ATOM 2284 CB ASP B 8 −4.203 18.473 22.030 1.00 8.76 C ATOM 2287 CG ASP B 8 −4.155 17.842 20.692 1.00 9.13 C ATOM 2288 OD1 ASP B 8 −3.323 18.263 19.835 1.00 6.14 O ATOM 2289 OD2 ASP B 8 −4.933 16.909 20.455 1.00 9.55 O ATOM 2290 C ASP B 8 −2.934 19.510 23.798 1.00 10.29 C ATOM 2291 O ASP B 8 −2.685 20.697 23.630 1.00 11.56 O ATOM 2292 N GLY B 9 −3.087 18.980 25.007 1.00 7.29 N ATOM 2294 CA GLY B 9 −3.174 19.910 26.104 1.00 8.84 C ATOM 2297 C GLY B 9 −1.801 20.412 26.478 1.00 9.08 C ATOM 2298 O GLY B 9 −1.625 21.602 26.761 1.00 8.49 O ATOM 2299 N VAL B 10 −0.795 19.565 26.363 1.00 10.58 N ATOM 2301 CA VAL B 10 0.591 20.016 26.620 1.00 8.31 C ATOM 2303 CB VAL B 10 1.507 18.863 27.007 1.00 9.15 C ATOM 2305 CG1 VAL B 10 2.903 19.389 27.165 1.00 11.22 C ATOM 2309 CG2 VAL B 10 1.009 18.122 28.215 1.00 11.83 C ATOM 2313 C VAL B 10 1.040 20.954 25.527 1.00 9.51 C ATOM 2314 O VAL B 10 1.647 21.983 25.807 1.00 9.75 O ATOM 2315 N ALA B 11 0.772 20.592 24.273 1.00 7.97 N ATOM 2317 CA ALA B 11 1.113 21.452 23.161 1.00 9.12 C ATOM 2319 CB ALA B 11 0.636 20.894 21.831 1.00 9.88 C ATOM 2323 C ALA B 11 0.580 22.870 23.301 1.00 7.14 C ATOM 2324 O ALA B 11 1.305 23.856 23.113 1.00 9.85 O ATOM 2325 N ASP B 12 −0.691 22.975 23.599 1.00 9.02 N ATOM 2327 CA ASP B 12 −1.289 24.252 23.744 1.00 10.56 C ATOM 2329 CB ASP B 12 −2.792 24.074 24.009 1.00 8.87 C ATOM 2332 CG ASP B 12 −3.522 23.540 22.769 1.00 9.50 C ATOM 2333 OD1 ASP B 12 −2.930 23.532 21.660 1.00 7.65 O ATOM 2334 OD2 ASP B 12 −4.768 23.257 22.802 1.00 9.59 O ATOM 2335 C ASP B 12 −0.679 25.010 24.937 1.00 9.51 C ATOM 2336 O ASP B 12 −0.483 26.212 24.813 1.00 13.07 O ATOM 2337 N TYR B 13 −0.468 24.333 26.037 1.00 9.80 N ATOM 2339 CA TYR B 13 0.066 24.973 27.238 1.00 7.80 C ATOM 2341 CB TYR B 13 0.160 24.034 28.450 1.00 7.41 C ATOM 2344 CG TYR B 13 0.391 24.735 29.769 1.00 8.08 C ATOM 2345 CD1 TYR B 13 1.654 24.907 30.268 1.00 9.81 C ATOM 2347 CE1 TYR B 13 1.923 25.538 31.426 1.00 9.09 C ATOM 2349 CZ TYR B 13 0.882 25.868 32.302 1.00 13.91 C ATOM 2350 OH TYR B 13 1.212 26.448 33.525 1.00 14.39 O ATOM 2352 CE2 TYR B 13 −0.458 25.581 31.903 1.00 10.49 C ATOM 2354 CD2 TYR B 13 −0.675 25.032 30.614 1.00 10.89 C ATOM 2356 C TYR B 13 1.483 25.494 26.973 1.00 7.88 C ATOM 2357 O TYR B 13 1.824 26.617 27.388 1.00 8.81 O ATOM 2358 N LEU B 14 2.309 24.624 26.447 1.00 10.94 N ATOM 2360 CA LEU B 14 3.638 25.014 25.990 1.00 7.43 C ATOM 2362 CB LEU B 14 4.233 23.858 25.220 1.00 9.68 C ATOM 2365 CG LEU B 14 4.871 22.796 26.149 1.00 10.03 C ATOM 2367 CD1 LEU B 14 5.122 21.584 25.320 1.00 10.85 C ATOM 2371 CD2 LEU B 14 6.178 23.145 26.903 1.00 9.85 C ATOM 2375 C LEU B 14 3.652 26.252 25.101 1.00 11.17 C ATOM 2376 O LEU B 14 4.475 27.159 25.320 1.00 10.57 O ATOM 2377 N GLN B 15 2.822 26.232 24.039 1.00 9.73 N ATOM 2379 CA GLN B 15 2.747 27.313 23.081 1.00 10.80 C ATOM 2381 CB GLN B 15 1.999 26.859 21.797 1.00 8.56 C ATOM 2384 CG GLN B 15 2.875 25.924 20.916 1.00 10.28 C ATOM 2387 CD GLN B 15 2.426 25.805 19.520 1.00 8.91 C ATOM 2388 OE1 GLN B 15 1.240 25.938 19.325 1.00 9.29 O ATOM 2389 NE2 GLN B 15 3.323 25.676 18.500 1.00 3.81 N ATOM 2392 C GLN B 15 2.186 28.597 23.639 1.00 10.08 C ATOM 2393 O GLN B 15 2.541 29.651 23.182 1.00 11.20 O ATOM 2394 N THR B 16 1.380 28.476 24.676 1.00 9.09 N ATOM 2396 CA THR B 16 0.794 29.547 25.357 1.00 9.13 C ATOM 2398 CB THR B 16 −0.534 29.194 25.979 1.00 7.00 C ATOM 2400 OG1 THR B 16 −1.481 28.772 25.021 1.00 10.11 O ATOM 2402 CG2 THR B 16 −1.142 30.399 26.664 1.00 8.72 C ATOM 2406 C THR B 16 1.736 30.206 26.395 1.00 7.73 C ATOM 2407 O THR B 16 1.955 31.388 26.349 1.00 6.63 O ATOM 2408 N TYR B 17 2.408 29.398 27.172 1.00 10.27 N ATOM 2410 CA TYR B 17 3.082 29.809 28.377 1.00 11.55 C ATOM 2412 CB TYR B 17 2.524 29.271 29.697 1.00 11.67 C ATOM 2415 CG TYR B 17 1.052 29.571 29.949 1.00 11.49 C ATOM 2416 CD1 TYR B 17 0.063 28.567 29.778 1.00 12.82 C ATOM 2418 CE1 TYR B 17 −1.313 28.791 30.003 1.00 11.99 C ATOM 2420 CZ TYR B 17 −1.685 30.091 30.334 1.00 12.14 C ATOM 2421 OH TYR B 17 −2.996 30.316 30.522 1.00 12.20 O ATOM 2423 CE2 TYR B 17 −0.737 31.122 30.513 1.00 12.43 C ATOM 2425 CD2 TYR B 17 0.635 30.828 30.373 1.00 11.40 C ATOM 2427 C TYR B 17 4.538 29.590 28.297 1.00 11.47 C ATOM 2428 O TYR B 17 5.234 30.053 29.222 1.00 11.44 O ATOM 2429 N HIS B 18 5.004 28.864 27.292 1.00 12.19 N ATOM 2431 CA HIS B 18 6.454 28.628 27.117 1.00 14.05 C ATOM 2433 CB HIS B 18 7.244 29.935 26.791 1.00 17.67 C ATOM 2436 CG HIS B 18 6.897 30.535 25.469 1.00 24.43 C ATOM 2437 ND1 HIS B 18 5.709 31.119 25.214 1.00 36.04 N ATOM 2439 CE1 HIS B 18 5.598 31.493 23.944 1.00 34.79 C ATOM 2441 NE2 HIS B 18 6.714 31.244 23.366 1.00 25.22 N ATOM 2443 CD2 HIS B 18 7.500 30.600 24.282 1.00 30.50 C ATOM 2445 C HIS B 18 7.135 27.925 28.303 1.00 13.17 C ATOM 2446 O HIS B 18 8.278 28.237 28.732 1.00 9.99 O ATOM 2447 N LYS B 19 6.359 27.047 28.905 1.00 11.45 N ATOM 2449 CA LYS B 19 6.879 26.163 29.940 1.00 15.06 C ATOM 2451 CB LYS B 19 6.989 26.910 31.307 1.00 15.74 C ATOM 2454 CG LYS B 19 5.637 27.434 31.872 1.00 18.65 C ATOM 2457 CD LYS B 19 5.833 28.453 33.009 1.00 21.85 C ATOM 2460 CE LYS B 19 4.569 28.960 33.644 1.00 24.72 C ATOM 2463 NZ LYS B 19 4.799 29.547 35.077 1.00 28.63 N ATOM 2467 C LYS B 19 5.897 25.010 30.086 1.00 12.64 C ATOM 2468 O LYS B 19 4.781 25.064 29.648 1.00 13.23 O ATOM 2469 N LEU B 20 6.335 23.888 30.616 1.00 13.20 N ATOM 2471 CA LEU B 20 5.414 22.809 30.925 1.00 10.09 C ATOM 2473 CB LEU B 20 6.204 21.566 31.307 1.00 11.80 C ATOM 2476 CG LEU B 20 7.128 21.060 30.215 1.00 11.10 C ATOM 2478 CD1 LEU B 20 8.164 20.025 30.597 1.00 13.97 C ATOM 2482 CD2 LEU B 20 6.205 20.302 29.184 1.00 12.08 C ATOM 2486 C LEU B 20 4.530 23.131 32.047 1.00 10.11 C ATOM 2487 O LEU B 20 4.832 23.921 32.929 1.00 11.78 O ATOM 2488 N PRO B 21 3.380 22.493 32.024 1.00 11.42 N ATOM 2489 CA PRO B 21 2.484 22.546 33.162 1.00 11.59 C ATOM 2491 CB PRO B 21 1.421 21.465 32.816 1.00 13.68 C ATOM 2494 CG PRO B 21 1.603 21.065 31.465 1.00 15.49 C ATOM 2497 CD PRO B 21 2.840 21.697 30.918 1.00 12.52 C ATOM 2500 C PRO B 21 3.197 22.133 34.490 1.00 11.12 C ATOM 2501 O PRO B 21 4.193 21.367 34.474 1.00 9.47 O ATOM 2502 N ASP B 22 2.542 22.447 35.582 1.00 10.82 N ATOM 2504 CA ASP B 22 3.111 22.219 36.896 1.00 16.29 C ATOM 2506 CB ASP B 22 2.227 22.925 37.937 1.00 19.70 C ATOM 2509 CG ASP B 22 2.415 24.383 37.824 1.00 25.42 C ATOM 2510 OD1 ASP B 22 1.646 25.091 38.504 1.00 28.81 O ATOM 2511 OD2 ASP B 22 3.315 24.931 37.119 1.00 25.63 O ATOM 2512 C ASP B 22 3.273 20.786 37.356 1.00 15.34 C ATOM 2513 O ASP B 22 3.754 20.552 38.484 1.00 16.59 O ATOM 2514 N ASN B 23 2.590 19.898 36.633 1.00 13.56 N ATOM 2516 CA ASN B 23 2.646 18.495 36.907 1.00 11.91 C ATOM 2518 CB ASN B 23 1.416 17.759 36.509 1.00 12.29 C ATOM 2521 CG ASN B 23 1.057 17.932 35.001 1.00 13.95 C ATOM 2522 OD1 ASN B 23 1.049 16.967 34.196 1.00 14.77 O ATOM 2523 ND2 ASN B 23 0.582 19.128 34.684 1.00 14.82 N ATOM 2526 C ASN B 23 3.849 17.836 36.358 1.00 11.72 C ATOM 2527 O ASN B 23 3.966 16.596 36.447 1.00 14.87 O ATOM 2528 N TYR B 24 4.781 18.628 35.847 1.00 10.27 N ATOM 2530 CA TYR B 24 5.980 18.036 35.240 1.00 10.75 C ATOM 2532 CB TYR B 24 6.294 18.529 33.842 1.00 10.64 C ATOM 2535 CG TYR B 24 5.428 17.929 32.790 1.00 10.75 C ATOM 2536 CD1 TYR B 24 5.774 16.719 32.234 1.00 9.86 C ATOM 2538 CE1 TYR B 24 4.897 16.041 31.364 1.00 10.65 C ATOM 2540 CZ TYR B 24 3.750 16.640 31.010 1.00 9.17 C ATOM 2541 OH TYR B 24 2.926 16.023 30.114 1.00 10.54 O ATOM 2543 CE2 TYR B 24 3.393 17.873 31.556 1.00 7.66 C ATOM 2545 CD2 TYR B 24 4.211 18.537 32.448 1.00 7.74 C ATOM 2547 C TYR B 24 7.237 18.279 36.101 1.00 11.32 C ATOM 2548 O TYR B 24 7.282 19.342 36.700 1.00 12.80 O ATOM 2549 N ILE B 25 8.156 17.313 36.205 1.00 11.07 N ATOM 2551 CA ILE B 25 9.469 17.534 36.882 1.00 11.11 C ATOM 2553 CB ILE B 25 9.525 16.893 38.258 1.00 12.23 C ATOM 2555 CG1 ILE B 25 9.303 15.401 38.162 1.00 12.51 C ATOM 2558 CD1 ILE B 25 9.362 14.819 39.651 1.00 13.73 C ATOM 2562 CG2 ILE B 25 8.615 17.678 39.189 1.00 14.66 C ATOM 2566 C ILE B 25 10.516 16.855 36.018 1.00 11.19 C ATOM 2567 O ILE B 25 10.145 15.932 35.223 1.00 12.24 O ATOM 2568 N THR B 26 11.731 17.382 36.073 1.00 11.68 N ATOM 2570 CA THR B 26 12.787 16.862 35.229 1.00 13.65 C ATOM 2572 CB THR B 26 14.052 17.739 35.301 1.00 14.60 C ATOM 2574 OG1 THR B 26 14.575 17.871 36.623 1.00 11.50 O ATOM 2576 CG2 THR B 26 13.809 19.193 34.703 1.00 15.85 C ATOM 2580 C THR B 26 13.192 15.474 35.761 1.00 14.64 C ATOM 2581 O THR B 26 12.920 15.113 36.936 1.00 12.25 O ATOM 2582 N LYS B 27 14.006 14.793 34.963 1.00 11.43 N ATOM 2584 CA LYS B 27 14.638 13.598 35.489 1.00 12.47 C ATOM 2586 CB LYS B 27 15.419 12.810 34.447 1.00 14.64 C ATOM 2589 CG LYS B 27 14.517 12.122 33.412 1.00 18.35 C ATOM 2592 CD LYS B 27 15.265 11.145 32.477 1.00 19.42 C ATOM 2595 CE LYS B 27 16.129 11.984 31.542 1.00 21.43 C ATOM 2598 NZ LYS B 27 15.335 12.839 30.547 1.00 18.82 N ATOM 2602 C LYS B 27 15.473 13.808 36.707 1.00 11.01 C ATOM 2603 O LYS B 27 15.316 13.002 37.640 1.00 9.61 O ATOM 2604 N SER B 28 16.387 14.758 36.704 1.00 10.20 N ATOM 2606 CA SER B 28 17.128 15.013 37.953 1.00 11.99 C ATOM 2608 CB SER B 28 18.136 16.166 38.038 1.00 14.58 C ATOM 2611 OG SER B 28 18.647 16.119 36.754 1.00 19.83 O ATOM 2613 C SER B 28 16.234 15.291 39.135 1.00 11.22 C ATOM 2614 O SER B 28 16.524 14.831 40.264 1.00 10.00 O ATOM 2615 N GLU B 29 15.239 16.103 38.896 1.00 10.23 N ATOM 2617 CA GLU B 29 14.316 16.394 39.989 1.00 11.87 C ATOM 2619 CB GLU B 29 13.293 17.461 39.653 1.00 15.30 C ATOM 2622 CG GLU B 29 13.879 18.873 39.597 1.00 17.38 C ATOM 2625 CD GLU B 29 12.942 19.806 38.872 1.00 19.55 C ATOM 2626 OE1 GLU B 29 13.272 21.005 38.905 1.00 23.69 O ATOM 2627 OE2 GLU B 29 11.953 19.376 38.212 1.00 19.38 O ATOM 2628 C GLU B 29 13.540 15.156 40.499 1.00 10.57 C ATOM 2629 O GLU B 29 13.466 15.001 41.690 1.00 10.87 O ATOM 2630 N ALA B 30 13.102 14.234 39.664 1.00 8.31 N ATOM 2632 CA ALA B 30 12.557 12.968 40.051 1.00 10.34 C ATOM 2634 CB ALA B 30 11.966 12.319 38.860 1.00 10.98 C ATOM 2638 C ALA B 30 13.604 12.060 40.741 1.00 10.90 C ATOM 2639 O ALA B 30 13.222 11.576 41.816 1.00 10.18 O ATOM 2640 N GLN B 31 14.870 11.974 40.286 1.00 12.79 N ATOM 2642 CA GLN B 31 15.934 11.215 40.895 1.00 14.39 C ATOM 2644 CB GLN B 31 17.277 11.212 40.156 1.00 19.96 C ATOM 2647 CG GLN B 31 17.216 10.729 38.640 1.00 24.22 C ATOM 2650 CD GLN B 31 18.537 10.673 37.723 1.00 28.13 C ATOM 2651 OE1 GLN B 31 19.059 11.660 37.122 1.00 29.70 O ATOM 2652 NE2 GLN B 31 18.981 9.434 37.520 1.00 28.68 N ATOM 2655 C GLN B 31 16.083 11.788 42.279 1.00 13.00 C ATOM 2656 O GLN B 31 16.267 10.994 43.172 1.00 11.92 O ATOM 2657 N ALA B 32 15.955 13.100 42.484 1.00 12.98 N ATOM 2659 CA ALA B 32 16.197 13.648 43.825 1.00 13.91 C ATOM 2661 CB ALA B 32 16.409 15.140 43.846 1.00 15.00 C ATOM 2665 C ALA B 32 15.136 13.268 44.853 1.00 13.28 C ATOM 2666 O ALA B 32 15.340 13.205 46.067 1.00 14.55 O ATOM 2667 N LEU B 33 13.983 12.906 44.331 1.00 13.14 N ATOM 2669 CA LEU B 33 12.881 12.507 45.171 1.00 12.01 C ATOM 2671 CB LEU B 33 11.565 12.938 44.511 1.00 13.86 C ATOM 2674 CG LEU B 33 11.274 14.421 44.500 1.00 15.58 C ATOM 2676 CD1 LEU B 33 10.166 14.775 43.483 1.00 15.99 C ATOM 2680 CD2 LEU B 33 10.908 14.822 46.002 1.00 17.33 C ATOM 2684 C LEU B 33 12.903 11.024 45.362 1.00 12.31 C ATOM 2685 O LEU B 33 12.048 10.518 46.123 1.00 11.39 O ATOM 2686 N GLY B 34 13.843 10.325 44.702 1.00 10.75 N ATOM 2688 CA GLY B 34 14.004 8.877 44.841 1.00 11.57 C ATOM 2691 C GLY B 34 13.539 7.995 43.709 1.00 13.21 C ATOM 2692 O GLY B 34 13.566 6.772 43.704 1.00 11.37 O ATOM 2693 N TRP B 35 13.097 8.673 42.658 1.00 12.54 N ATOM 2695 CA TRP B 35 12.875 8.003 41.406 1.00 14.11 C ATOM 2697 CB TRP B 35 12.401 8.916 40.272 1.00 13.66 C ATOM 2700 CG TRP B 35 12.205 8.174 38.906 1.00 13.56 C ATOM 2701 CD1 TRP B 35 11.337 7.122 38.634 1.00 15.31 C ATOM 2703 NE1 TRP B 35 11.394 6.748 37.311 1.00 13.91 N ATOM 2705 CE2 TRP B 35 12.381 7.499 36.720 1.00 14.38 C ATOM 2706 CD2 TRP B 35 12.925 8.375 37.714 1.00 13.95 C ATOM 2707 CE3 TRP B 35 13.899 9.305 37.322 1.00 14.84 C ATOM 2709 CZ3 TRP B 35 14.410 9.260 36.029 1.00 13.69 C ATOM 2711 CH2 TRP B 35 13.893 8.324 35.067 1.00 13.19 C ATOM 2713 CZ2 TRP B 35 12.887 7.448 35.416 1.00 14.36 C ATOM 2715 C TRP B 35 14.115 7.243 40.896 1.00 14.04 C ATOM 2716 O TRP B 35 15.190 7.801 40.716 1.00 15.29 O ATOM 2717 N VAL B 36 13.881 6.010 40.477 1.00 13.88 N ATOM 2719 CA VAL B 36 14.938 5.224 39.851 1.00 15.14 C ATOM 2721 CB VAL B 36 15.528 4.160 40.879 1.00 16.48 C ATOM 2723 CG1 VAL B 36 16.548 3.157 40.227 1.00 16.99 C ATOM 2727 CG2 VAL B 36 16.171 4.808 42.113 1.00 17.44 C ATOM 2731 C VAL B 36 14.267 4.521 38.670 1.00 14.82 C ATOM 2732 O VAL B 36 13.314 3.811 38.897 1.00 12.15 O ATOM 2733 N ALA B 37 14.782 4.608 37.446 1.00 16.44 N ATOM 2735 CA ALA B 37 14.068 3.997 36.306 1.00 18.37 C ATOM 2737 CB ALA B 37 14.815 4.224 35.074 1.00 18.92 C ATOM 2741 C ALA B 37 13.684 2.542 36.399 1.00 20.89 C ATOM 2742 O ALA B 37 12.480 2.155 36.213 1.00 24.01 O ATOM 2743 N SER B 38 14.664 1.698 36.699 1.00 22.10 N ATOM 2745 CA SER B 38 14.283 0.259 36.837 1.00 22.69 C ATOM 2747 CB SER B 38 15.559 −0.554 37.100 1.00 21.57 C ATOM 2750 OG SER B 38 16.227 −0.185 38.313 1.00 22.63 O ATOM 2752 C SER B 38 13.211 −0.060 37.961 1.00 22.94 C ATOM 2753 O SER B 38 12.833 −1.217 38.223 1.00 22.63 O ATOM 2754 N LYS B 39 12.802 0.929 38.751 1.00 20.18 N ATOM 2756 CA LYS B 39 11.882 0.673 39.846 1.00 20.50 C ATOM 2758 CB LYS B 39 12.320 1.320 41.183 1.00 21.23 C ATOM 2761 CG LYS B 39 13.561 0.765 41.893 1.00 24.77 C ATOM 2764 CD LYS B 39 13.538 1.201 43.423 1.00 28.33 C ATOM 2767 CE LYS B 39 14.880 1.154 44.242 1.00 29.68 C ATOM 2770 NZ LYS B 39 15.134 2.247 45.316 1.00 28.89 N ATOM 2774 C LYS B 39 10.403 0.992 39.492 1.00 17.00 C ATOM 2775 O LYS B 39 9.506 0.664 40.253 1.00 13.44 O ATOM 2776 N GLY B 40 10.178 1.681 38.387 1.00 14.62 N ATOM 2778 CA GLY B 40 8.861 2.155 37.995 1.00 13.91 C ATOM 2781 C GLY B 40 8.100 2.925 39.085 1.00 14.90 C ATOM 2782 O GLY B 40 6.841 2.864 39.180 1.00 13.49 O ATOM 2783 N ASN B 41 8.846 3.662 39.919 1.00 12.54 N ATOM 2785 CA ASN B 41 8.311 4.283 41.124 1.00 12.94 C ATOM 2787 CB ASN B 41 9.245 3.914 42.277 1.00 12.00 C ATOM 2790 CG ASN B 41 10.602 4.641 42.160 1.00 11.23 C ATOM 2791 OD1 ASN B 41 11.063 5.011 41.079 1.00 10.63 O ATOM 2792 ND2 ASN B 41 11.235 4.830 43.312 1.00 10.22 N ATOM 2795 C ASN B 41 8.030 5.836 41.123 1.00 11.22 C ATOM 2796 O ASN B 41 8.079 6.456 42.186 1.00 13.26 O ATOM 2797 N LEU B 42 7.855 6.431 39.942 1.00 13.90 N ATOM 2799 CA LEU B 42 7.820 7.901 39.808 1.00 13.55 C ATOM 2801 CB LEU B 42 7.718 8.373 38.389 1.00 14.14 C ATOM 2804 CG LEU B 42 7.483 9.856 38.161 1.00 13.84 C ATOM 2806 CD1 LEU B 42 8.631 10.589 38.839 1.00 14.77 C ATOM 2810 CD2 LEU B 42 7.361 10.199 36.694 1.00 15.89 C ATOM 2814 C LEU B 42 6.595 8.359 40.621 1.00 12.12 C ATOM 2815 O LEU B 42 6.728 9.265 41.412 1.00 11.43 O ATOM 2816 N ALA B 43 5.458 7.694 40.471 1.00 15.11 N ATOM 2818 CA ALA B 43 4.210 8.094 41.091 1.00 16.61 C ATOM 2820 CB ALA B 43 3.000 7.567 40.368 1.00 16.87 C ATOM 2824 C ALA B 43 4.147 7.827 42.567 1.00 16.65 C ATOM 2825 O ALA B 43 3.317 8.440 43.242 1.00 15.47 O ATOM 2826 N ASP B 44 5.027 6.965 43.055 1.00 18.19 N ATOM 2828 CA ASP B 44 5.135 6.790 44.497 1.00 19.89 C ATOM 2830 CB ASP B 44 5.853 5.480 44.884 1.00 24.16 C ATOM 2833 CG ASP B 44 5.218 4.261 44.280 1.00 27.36 C ATOM 2834 OD1 ASP B 44 4.018 4.065 44.502 1.00 30.60 O ATOM 2835 OD2 ASP B 44 5.786 3.464 43.507 1.00 30.35 O ATOM 2836 C ASP B 44 5.942 7.921 45.115 1.00 17.87 C ATOM 2837 O ASP B 44 5.640 8.343 46.217 1.00 18.04 O ATOM 2838 N VAL B 45 6.999 8.399 44.440 1.00 15.56 N ATOM 2840 CA VAL B 45 7.785 9.445 45.099 1.00 12.84 C ATOM 2842 CB VAL B 45 9.255 9.309 44.795 1.00 14.45 C ATOM 2844 CG1 VAL B 45 9.727 7.915 45.156 1.00 16.75 C ATOM 2848 CG2 VAL B 45 9.571 9.462 43.280 1.00 13.91 C ATOM 2852 C VAL B 45 7.389 10.830 44.658 1.00 12.95 C ATOM 2853 O VAL B 45 7.815 11.800 45.278 1.00 12.61 O ATOM 2854 N ALA B 46 6.672 10.937 43.529 1.00 11.48 N ATOM 2856 CA ALA B 46 6.202 12.235 43.022 1.00 13.03 C ATOM 2858 CB ALA B 46 7.091 12.748 41.873 1.00 12.22 C ATOM 2862 C ALA B 46 4.760 12.084 42.499 1.00 11.88 C ATOM 2863 O ALA B 46 4.468 12.032 41.324 1.00 14.30 O ATOM 2864 N PRO B 47 3.838 12.052 43.426 1.00 13.53 N ATOM 2865 CA PRO B 47 2.496 11.645 43.052 1.00 14.04 C ATOM 2867 CB PRO B 47 1.741 11.717 44.404 1.00 14.16 C ATOM 2870 CG PRO B 47 2.822 11.473 45.404 1.00 15.19 C ATOM 2873 CD PRO B 47 4.046 12.158 44.894 1.00 15.11 C ATOM 2876 C PRO B 47 1.920 12.675 42.107 1.00 13.57 C ATOM 2877 O PRO B 47 2.089 13.855 42.339 1.00 14.43 O ATOM 2878 N GLY B 48 1.333 12.257 40.997 1.00 13.92 N ATOM 2880 CA GLY B 48 0.699 13.206 40.107 1.00 11.07 C ATOM 2883 C GLY B 48 1.615 13.814 39.081 1.00 12.05 C ATOM 2884 O GLY B 48 1.148 14.637 38.248 1.00 14.31 O ATOM 2885 N LYS B 49 2.915 13.550 39.263 1.00 9.92 N ATOM 2887 CA LYS B 49 3.885 14.055 38.328 1.00 9.15 C ATOM 2889 CB LYS B 49 5.107 14.440 39.118 1.00 8.77 C ATOM 2892 CG LYS B 49 4.764 15.397 40.227 1.00 10.89 C ATOM 2895 CD LYS B 49 4.328 16.788 39.963 1.00 13.20 C ATOM 2898 CE LYS B 49 4.423 17.615 41.294 1.00 15.89 C ATOM 2901 NZ LYS B 49 4.288 18.964 40.734 1.00 18.50 N ATOM 2905 C LYS B 49 4.295 13.129 37.237 1.00 11.64 C ATOM 2906 O LYS B 49 3.919 11.969 37.234 1.00 11.93 O ATOM 2907 N SER B 50 4.798 13.760 36.175 1.00 13.66 N ATOM 2909 CA SER B 50 5.296 13.128 34.984 1.00 12.63 C ATOM 2911 CB SER B 50 4.473 13.411 33.703 1.00 12.14 C ATOM 2914 OG SER B 50 3.118 12.953 33.793 1.00 9.72 O ATOM 2916 C SER B 50 6.698 13.748 34.777 1.00 13.09 C ATOM 2917 O SER B 50 6.917 14.924 35.064 1.00 10.65 O ATOM 2918 N ILE B 51 7.584 12.935 34.212 1.00 10.44 N ATOM 2920 CA ILE B 51 8.850 13.427 33.834 1.00 11.52 C ATOM 2922 CB ILE B 51 9.737 12.216 33.416 1.00 13.32 C ATOM 2924 CG1 ILE B 51 10.153 11.244 34.558 1.00 13.81 C ATOM 2927 CD1 ILE B 51 10.718 12.028 35.481 1.00 15.55 C ATOM 2931 CG2 ILE B 51 10.996 12.720 32.766 1.00 9.34 C ATOM 2935 C ILE B 51 8.731 14.313 32.525 1.00 12.05 C ATOM 2936 O ILE B 51 8.047 14.016 31.561 1.00 12.48 O ATOM 2937 N GLY B 52 9.345 15.474 32.534 1.00 12.14 N ATOM 2939 CA GLY B 52 9.413 16.324 31.357 1.00 11.66 C ATOM 2942 C GLY B 52 10.335 17.490 31.576 1.00 12.77 C ATOM 2943 O GLY B 52 10.646 17.884 32.723 1.00 10.85 O ATOM 2944 N GLY B 53 10.697 18.089 30.445 1.00 10.59 N ATOM 2946 CA GLY B 53 11.507 19.295 30.437 1.00 13.12 C ATOM 2949 C GLY B 53 12.956 19.111 30.057 1.00 13.01 C ATOM 2950 O GLY B 53 13.711 20.073 29.916 1.00 12.93 O ATOM 2951 N ASP B 54 13.396 17.870 30.032 1.00 11.51 N ATOM 2953 CA ASP B 54 14.787 17.675 29.793 1.00 13.36 C ATOM 2955 CB ASP B 54 15.291 16.320 30.214 1.00 14.97 C ATOM 2958 CG ASP B 54 15.028 16.059 31.664 1.00 17.99 C ATOM 2959 OD1 ASP B 54 15.442 16.921 32.461 1.00 17.16 O ATOM 2960 OD2 ASP B 54 14.309 15.092 32.022 1.00 16.58 O ATOM 2961 C ASP B 54 15.066 17.807 28.306 1.00 14.77 C ATOM 2962 O ASP B 54 14.227 17.691 27.416 1.00 12.24 O ATOM 2963 N ILE B 55 16.316 18.144 28.050 1.00 13.38 N ATOM 2965 CA ILE B 55 16.798 18.205 26.667 1.00 13.79 C ATOM 2967 CB ILE B 55 18.301 18.698 26.674 1.00 13.55 C ATOM 2969 CG1 ILE B 55 18.318 20.169 27.096 1.00 14.46 C ATOM 2972 CD1 ILE B 55 18.098 21.020 25.908 1.00 17.36 C ATOM 2976 CG2 ILE B 55 19.033 18.502 25.271 1.00 13.32 C ATOM 2980 C ILE B 55 16.757 16.887 25.926 1.00 13.67 C ATOM 2981 O ILE B 55 17.117 15.856 26.515 1.00 11.34 O ATOM 2982 N PHE B 56 16.372 16.956 24.652 1.00 12.37 N ATOM 2984 CA PHE B 56 16.420 15.781 23.781 1.00 14.85 C ATOM 2986 CB PHE B 56 15.066 15.540 23.131 1.00 14.98 C ATOM 2989 CG PHE B 56 15.045 14.462 22.122 1.00 14.15 C ATOM 2990 CD1 PHE B 56 15.094 13.163 22.492 1.00 16.41 C ATOM 2992 CE1 PHE B 56 15.080 12.101 21.579 1.00 15.50 C ATOM 2994 CZ PHE B 56 14.954 12.359 20.275 1.00 17.62 C ATOM 2996 CE2 PHE B 56 14.937 13.684 19.815 1.00 17.52 C ATOM 2998 CD2 PHE B 56 14.927 14.754 20.797 1.00 17.22 C ATOM 3000 C PHE B 56 17.495 16.046 22.751 1.00 16.47 C ATOM 3001 O PHE B 56 17.319 16.988 21.993 1.00 17.74 O ATOM 3002 N SER B 57 18.612 15.311 22.739 1.00 16.03 N ATOM 3004 CA SER B 57 19.710 15.647 21.856 1.00 19.63 C ATOM 3006 CB SER B 57 20.865 14.712 22.157 1.00 21.92 C ATOM 3009 OG SER B 57 20.198 13.480 21.883 1.00 28.28 O ATOM 3011 C SER B 57 19.377 15.424 20.380 1.00 19.52 C ATOM 3012 O SER B 57 20.000 16.032 19.536 1.00 19.22 O ATOM 3013 N ASN B 58 18.403 14.613 19.985 1.00 19.47 N ATOM 3015 CA ASN B 58 18.061 14.614 18.532 1.00 19.30 C ATOM 3017 CB ASN B 58 17.625 15.998 17.965 1.00 16.59 C ATOM 3020 CG ASN B 58 16.947 15.904 16.623 1.00 14.55 C ATOM 3021 OD1 ASN B 58 16.192 14.958 16.347 1.00 10.91 O ATOM 3022 ND2 ASN B 58 17.143 16.939 15.797 1.00 13.94 N ATOM 3025 C ASN B 58 19.192 14.060 17.696 1.00 21.21 C ATOM 3026 O ASN B 58 19.395 14.477 16.557 1.00 18.51 O ATOM 3027 N ARG B 59 19.879 13.089 18.291 1.00 25.14 N ATOM 3029 CA ARG B 59 21.127 12.526 17.763 1.00 29.09 C ATOM 3031 CB ARG B 59 21.680 11.481 18.820 1.00 33.53 C ATOM 3034 CG ARG B 59 22.910 11.914 19.812 1.00 37.93 C ATOM 3037 CD ARG B 59 23.254 11.163 21.216 1.00 40.80 C ATOM 3040 NE ARG B 59 22.131 10.714 22.100 1.00 43.15 N ATOM 3042 CZ ARG B 59 21.898 10.960 23.418 1.00 44.23 C ATOM 3043 NH1 ARG B 59 22.695 11.724 24.163 1.00 44.03 N ATOM 3046 NH2 ARG B 59 20.789 10.467 23.981 1.00 44.52 N ATOM 3049 C ARG B 59 20.826 11.953 16.339 1.00 27.38 C ATOM 3050 O ARG B 59 21.563 12.155 15.376 1.00 24.38 O ATOM 3051 N GLU B 60 19.714 11.230 16.180 1.00 26.08 N ATOM 3053 CA GLU B 60 19.389 10.662 14.876 1.00 25.44 C ATOM 3055 CB GLU B 60 18.314 9.610 15.037 1.00 28.90 C ATOM 3058 CG GLU B 60 18.542 8.639 16.184 1.00 33.14 C ATOM 3061 CD GLU B 60 17.717 7.366 16.041 1.00 36.08 C ATOM 3062 OE1 GLU B 60 16.456 7.363 16.154 1.00 38.14 O ATOM 3063 OE2 GLU B 60 18.366 6.340 15.766 1.00 40.88 O ATOM 3064 C GLU B 60 18.962 11.652 13.795 1.00 21.86 C ATOM 3065 O GLU B 60 18.647 11.282 12.668 1.00 20.52 O ATOM 3066 N GLY B 61 18.749 12.897 14.174 1.00 17.18 N ATOM 3068 CA GLY B 61 18.395 13.868 13.195 1.00 15.80 C ATOM 3071 C GLY B 61 16.964 13.696 12.721 1.00 14.53 C ATOM 3072 O GLY B 61 16.660 14.211 11.681 1.00 10.59 O ATOM 3073 N LYS B 62 16.111 12.944 13.380 1.00 13.39 N ATOM 3075 CA LYS B 62 14.725 12.841 12.896 1.00 16.33 C ATOM 3077 CB LYS B 62 14.036 11.690 13.652 1.00 17.63 C ATOM 3080 CG LYS B 62 14.428 10.354 13.017 1.00 19.36 C ATOM 3083 CD LYS B 62 14.161 9.185 14.019 1.00 19.12 C ATOM 3086 CE LYS B 62 14.686 7.875 13.322 1.00 19.49 C ATOM 3089 NZ LYS B 62 14.351 6.710 14.216 1.00 16.20 N ATOM 3093 C LYS B 62 13.829 14.062 13.083 1.00 16.26 C ATOM 3094 O LYS B 62 12.833 14.214 12.382 1.00 16.49 O ATOM 3095 N LEU B 63 14.135 14.915 14.054 1.00 13.74 N ATOM 3097 CA LEU B 63 13.442 16.167 14.120 1.00 14.01 C ATOM 3099 CB LEU B 63 13.322 16.685 15.525 1.00 13.34 C ATOM 3102 CG LEU B 63 12.510 15.779 16.426 1.00 14.81 C ATOM 3104 CD1 LEU B 63 12.665 16.074 17.911 1.00 14.43 C ATOM 3108 CD2 LEU B 63 10.991 15.726 15.995 1.00 14.13 C ATOM 3112 C LEU B 63 14.242 17.186 13.315 1.00 15.51 C ATOM 3113 O LEU B 63 15.480 17.144 13.327 1.00 10.44 O ATOM 3114 N PRO B 64 13.572 18.214 12.783 1.00 14.48 N ATOM 3115 CA PRO B 64 14.317 19.202 12.007 1.00 16.32 C ATOM 3117 CB PRO B 64 13.199 20.032 11.353 1.00 16.04 C ATOM 3120 CG PRO B 64 12.037 19.914 12.287 1.00 17.22 C ATOM 3123 CD PRO B 64 12.165 18.574 12.919 1.00 16.49 C ATOM 3126 C PRO B 64 15.157 20.030 12.963 1.00 15.25 C ATOM 3127 O PRO B 64 14.728 20.446 14.048 1.00 13.62 O ATOM 3128 N GLY B 65 16.358 20.297 12.528 1.00 14.61 N ATOM 3130 CA GLY B 65 17.285 21.088 13.297 1.00 15.69 C ATOM 3133 C GLY B 65 17.804 22.369 12.670 1.00 15.21 C ATOM 3134 O GLY B 65 17.764 22.459 11.472 1.00 14.39 O ATOM 3135 N LYS B 66 18.119 23.404 13.447 1.00 10.95 N ATOM 3137 CA LYS B 66 18.781 24.548 12.841 1.00 13.11 C ATOM 3139 CB LYS B 66 17.791 25.562 12.322 1.00 12.82 C ATOM 3142 CG LYS B 66 17.007 26.296 13.407 1.00 13.39 C ATOM 3145 CD LYS B 66 16.013 27.309 12.776 1.00 13.95 C ATOM 3148 CE LYS B 66 15.176 28.166 13.786 1.00 16.36 C ATOM 3151 NZ LYS B 66 14.165 29.115 13.158 1.00 11.77 N ATOM 3155 C LYS B 66 19.614 25.165 13.933 1.00 14.09 C ATOM 3156 O LYS B 66 19.343 24.994 15.142 1.00 12.00 O ATOM 3157 N SER B 67 20.616 25.912 13.509 1.00 14.90 N ATOM 3159 CA SER B 67 21.466 26.548 14.492 1.00 15.76 C ATOM 3161 CB SER B 67 22.546 27.250 13.640 1.00 20.81 C ATOM 3164 OG SER B 67 22.716 28.501 14.270 1.00 25.41 O ATOM 3166 C SER B 67 20.676 27.446 15.495 1.00 15.12 C ATOM 3167 O SER B 67 19.716 28.121 15.161 1.00 12.04 O ATOM 3168 N GLY B 68 21.006 27.309 16.780 1.00 14.59 N ATOM 3170 CA GLY B 68 20.321 27.923 17.881 1.00 15.20 C ATOM 3173 C GLY B 68 19.087 27.131 18.370 1.00 16.18 C ATOM 3174 O GLY B 68 18.508 27.506 19.432 1.00 16.23 O ATOM 3175 N ARG B 69 18.602 26.098 17.644 1.00 12.19 N ATOM 3177 CA ARG B 69 17.300 25.498 18.109 1.00 11.38 C ATOM 3179 CB ARG B 69 16.581 24.937 16.919 1.00 11.11 C ATOM 3182 CG ARG B 69 15.380 24.063 17.328 1.00 10.07 C ATOM 3185 CD ARG B 69 14.825 23.413 16.057 1.00 10.96 C ATOM 3188 NE ARG B 69 13.998 24.352 15.316 1.00 11.66 N ATOM 3190 CZ ARG B 69 13.644 24.264 14.049 1.00 11.42 C ATOM 3191 NH1 ARG B 69 14.074 23.229 13.293 1.00 13.20 N ATOM 3194 NH2 ARG B 69 12.965 25.285 13.515 1.00 12.11 N ATOM 3197 C ARG B 69 17.612 24.241 18.979 1.00 11.99 C ATOM 3198 O ARG B 69 18.424 23.428 18.567 1.00 12.40 O ATOM 3199 N THR B 70 17.037 24.125 20.169 1.00 13.86 N ATOM 3201 CA THR B 70 17.180 22.979 20.966 1.00 13.56 C ATOM 3203 CB THR B 70 17.851 23.241 22.332 1.00 15.52 C ATOM 3205 OG1 THR B 70 17.244 24.348 22.902 1.00 18.34 O ATOM 3207 CG2 THR B 70 19.246 23.871 22.193 1.00 14.51 C ATOM 3211 C THR B 70 15.853 22.374 21.154 1.00 14.51 C ATOM 3212 O THR B 70 14.787 23.010 21.002 1.00 13.92 O ATOM 3213 N TRP B 71 15.943 21.072 21.403 1.00 11.24 N ATOM 3215 CA TRP B 71 14.698 20.359 21.574 1.00 10.75 C ATOM 3217 CB TRP B 71 14.701 19.142 20.651 1.00 10.35 C ATOM 3220 CG TRP B 71 14.455 19.476 19.315 1.00 9.15 C ATOM 3221 CD1 TRP B 71 15.385 19.661 18.348 1.00 10.42 C ATOM 3223 NE1 TRP B 71 14.764 19.960 17.162 1.00 8.90 N ATOM 3225 CE2 TRP B 71 13.405 19.886 17.349 1.00 9.90 C ATOM 3226 CD2 TRP B 71 13.176 19.664 18.707 1.00 8.92 C ATOM 3227 CE3 TRP B 71 11.850 19.587 19.162 1.00 8.43 C ATOM 3229 CZ3 TRP B 71 10.798 19.818 18.226 1.00 9.38 C ATOM 3231 CH2 TRP B 71 11.090 20.155 16.909 1.00 10.00 C ATOM 3233 CZ2 TRP B 71 12.402 20.134 16.460 1.00 10.24 C ATOM 3235 C TRP B 71 14.522 19.900 23.013 1.00 11.11 C ATOM 3236 O TRP B 71 15.525 19.432 23.608 1.00 12.14 O ATOM 3237 N ARG B 72 13.281 19.824 23.522 1.00 8.93 N ATOM 3239 CA ARG B 72 13.148 19.254 24.831 1.00 8.99 C ATOM 3241 CB ARG B 72 12.781 20.436 25.772 1.00 10.59 C ATOM 3244 CG ARG B 72 14.049 21.172 26.206 1.00 10.47 C ATOM 3247 CD ARG B 72 13.698 22.350 27.185 1.00 13.16 C ATOM 3250 NE ARG B 72 14.829 23.234 27.457 1.00 17.14 N ATOM 3252 CZ ARG B 72 15.765 23.008 28.396 1.00 21.76 C ATOM 3253 NH1 ARG B 72 15.799 21.977 29.248 1.00 19.74 N ATOM 3256 NH2 ARG B 72 16.755 23.874 28.474 1.00 24.92 N ATOM 3259 C ARG B 72 11.981 18.317 24.711 1.00 11.55 C ATOM 3260 O ARG B 72 11.309 18.298 23.723 1.00 9.98 O ATOM 3261 N GLU B 73 11.805 17.467 25.705 1.00 11.24 N ATOM 3263 CA GLU B 73 10.800 16.416 25.686 1.00 11.94 C ATOM 3265 CB GLU B 73 11.461 15.059 25.499 1.00 11.99 C ATOM 3268 CG GLU B 73 12.550 14.639 26.507 1.00 12.70 C ATOM 3271 CD GLU B 73 12.781 13.157 26.547 1.00 13.39 C ATOM 3272 OE1 GLU B 73 11.866 12.343 26.858 1.00 14.56 O ATOM 3273 OE2 GLU B 73 13.883 12.799 26.099 1.00 15.23 O ATOM 3274 C GLU B 73 10.039 16.368 27.041 1.00 12.76 C ATOM 3275 O GLU B 73 10.496 16.946 28.045 1.00 10.99 O ATOM 3276 N ALA B 74 8.830 15.836 26.985 1.00 10.61 N ATOM 3278 CA ALA B 74 8.102 15.625 28.176 1.00 9.54 C ATOM 3280 CB ALA B 74 7.150 16.788 28.479 1.00 12.29 C ATOM 3284 C ALA B 74 7.222 14.372 27.940 1.00 10.03 C ATOM 3285 O ALA B 74 6.814 14.034 26.786 1.00 8.68 O ATOM 3286 N ASP B 75 6.938 13.688 29.038 1.00 10.21 N ATOM 3288 CA ASP B 75 6.139 12.457 28.976 1.00 10.00 C ATOM 3290 CB ASP B 75 6.316 11.625 30.287 1.00 10.62 C ATOM 3293 CG ASP B 75 7.582 10.826 30.281 1.00 11.53 C ATOM 3294 OD1 ASP B 75 8.535 11.138 29.497 1.00 10.00 O ATOM 3295 OD2 ASP B 75 7.689 9.813 31.005 1.00 8.42 O ATOM 3296 C ASP B 75 4.694 12.806 28.893 1.00 10.82 C ATOM 3297 O ASP B 75 4.156 13.698 29.619 1.00 10.82 O ATOM 3298 N ILE B 76 4.017 12.065 28.004 1.00 10.07 N ATOM 3300 CA ILE B 76 2.593 12.248 27.773 1.00 10.81 C ATOM 3302 CB ILE B 76 2.402 12.577 26.295 1.00 11.34 C ATOM 3304 CG1 ILE B 76 3.067 13.908 25.924 1.00 11.79 C ATOM 3307 CD1 ILE B 76 2.244 15.072 26.633 1.00 9.59 C ATOM 3311 CG2 ILE B 76 0.913 12.450 25.934 1.00 11.30 C ATOM 3315 C ILE B 76 1.899 10.939 28.162 1.00 11.44 C ATOM 3316 O ILE B 76 2.447 9.834 28.008 1.00 12.67 O ATOM 3317 N ASN B 77 0.671 11.137 28.634 1.00 11.47 N ATOM 3319 CA ASN B 77 −0.308 10.132 28.985 1.00 11.42 C ATOM 3321 CB ASN B 77 −0.718 9.193 27.842 1.00 10.13 C ATOM 3324 CG ASN B 77 −1.413 9.887 26.689 1.00 10.02 C ATOM 3325 OD1 ASN B 77 −2.277 10.816 26.849 1.00 10.14 O ATOM 3326 ND2 ASN B 77 −1.086 9.387 25.484 1.00 5.55 N ATOM 3329 C ASN B 77 0.203 9.321 30.171 1.00 10.23 C ATOM 3330 O ASN B 77 −0.293 8.237 30.365 1.00 8.49 O ATOM 3331 N TYR B 78 1.223 9.787 30.881 1.00 10.59 N ATOM 3333 CA TYR B 78 1.713 9.110 32.078 1.00 10.50 C ATOM 3335 CB TYR B 78 3.165 9.533 32.418 1.00 13.30 C ATOM 3338 CG TYR B 78 3.677 8.796 33.651 1.00 11.64 C ATOM 3339 CD1 TYR B 78 4.238 7.543 33.513 1.00 9.95 C ATOM 3341 CE1 TYR B 78 4.523 6.798 34.604 1.00 10.12 C ATOM 3343 CZ TYR B 78 4.536 7.399 35.866 1.00 13.58 C ATOM 3344 OH TYR B 78 4.921 6.680 37.002 1.00 11.86 O ATOM 3346 CE2 TYR B 78 4.018 8.680 36.038 1.00 13.35 C ATOM 3348 CD2 TYR B 78 3.609 9.363 34.905 1.00 13.98 C ATOM 3350 C TYR B 78 0.830 9.346 33.299 1.00 13.49 C ATOM 3351 O TYR B 78 0.502 10.501 33.576 1.00 12.46 O ATOM 3352 N THR B 79 0.501 8.269 34.029 1.00 15.53 N ATOM 3354 CA THR B 79 −0.174 8.403 35.311 1.00 16.93 C ATOM 3356 CB THR B 79 −1.630 7.862 35.308 1.00 19.69 C ATOM 3358 OG1 THR B 79 −1.566 6.566 34.809 1.00 22.41 O ATOM 3360 CG2 THR B 79 −2.524 8.426 34.198 1.00 20.10 C ATOM 3364 C THR B 79 0.608 7.691 36.442 1.00 16.57 C ATOM 3365 O THR B 79 0.936 8.345 37.437 1.00 17.72 O ATOM 3366 N SER B 80 1.012 6.438 36.273 1.00 14.71 N ATOM 3368 CA SER B 80 1.693 5.684 37.324 1.00 17.33 C ATOM 3370 CB SER B 80 0.768 5.182 38.478 1.00 17.59 C ATOM 3373 OG SER B 80 −0.117 4.227 37.922 1.00 16.70 O ATOM 3375 C SER B 80 2.403 4.483 36.751 1.00 14.92 C ATOM 3376 O SER B 80 2.137 4.104 35.610 1.00 16.85 O ATOM 3377 N GLY B 81 3.424 4.035 37.455 1.00 11.29 N ATOM 3379 CA GLY B 81 4.109 2.836 37.041 1.00 12.87 C ATOM 3382 C GLY B 81 5.316 3.161 36.204 1.00 12.63 C ATOM 3383 O GLY B 81 5.829 4.265 36.295 1.00 10.91 O ATOM 3384 N PHE B 82 5.668 2.273 35.289 1.00 14.08 N ATOM 3386 CA PHE B 82 6.868 2.483 34.496 1.00 15.62 C ATOM 3388 CB PHE B 82 7.451 1.210 33.794 1.00 16.18 C ATOM 3391 CG PHE B 82 8.098 0.281 34.762 1.00 16.15 C ATOM 3392 CD1 PHE B 82 9.459 0.374 35.054 1.00 16.99 C ATOM 3394 CE1 PHE B 82 9.997 −0.460 36.018 1.00 17.27 C ATOM 3396 CZ PHE B 82 9.161 −1.307 36.716 1.00 17.12 C ATOM 3398 CE2 PHE B 82 7.825 −1.460 36.320 1.00 16.78 C ATOM 3400 CD2 PHE B 82 7.305 −0.598 35.425 1.00 16.65 C ATOM 3402 C PHE B 82 6.430 3.554 33.479 1.00 15.75 C ATOM 3403 O PHE B 82 5.176 3.654 33.215 1.00 13.08 O ATOM 3404 N ARG B 83 7.447 4.202 32.872 1.00 14.73 N ATOM 3406 CA ARG B 83 7.168 5.264 31.864 1.00 14.03 C ATOM 3408 CB ARG B 83 8.423 6.104 31.539 1.00 14.15 C ATOM 3411 CG ARG B 83 8.896 6.789 32.802 1.00 12.57 C ATOM 3414 CD ARG B 83 10.241 7.476 32.590 1.00 12.77 C ATOM 3417 NE ARG B 83 10.149 8.493 31.525 1.00 12.31 N ATOM 3419 CZ ARG B 83 11.192 9.088 30.968 1.00 13.30 C ATOM 3420 NH1 ARG B 83 12.330 8.569 31.242 1.00 12.84 N ATOM 3423 NH2 ARG B 83 11.094 10.007 29.995 1.00 13.50 N ATOM 3426 C ARG B 83 6.746 4.523 30.603 1.00 14.98 C ATOM 3427 O ARG B 83 7.169 3.385 30.333 1.00 13.79 O ATOM 3428 N ASN B 84 5.827 5.172 29.929 1.00 13.06 N ATOM 3430 CA ASN B 84 5.339 4.760 28.643 1.00 11.13 C ATOM 3432 CB ASN B 84 3.862 5.136 28.564 1.00 9.88 C ATOM 3435 CG ASN B 84 3.674 6.630 28.364 1.00 9.84 C ATOM 3436 OD1 ASN B 84 4.576 7.309 27.879 1.00 7.46 O ATOM 3437 ND2 ASN B 84 2.519 7.159 28.767 1.00 6.81 N ATOM 3440 C ASN B 84 6.196 5.228 27.438 1.00 11.63 C ATOM 3441 O ASN B 84 7.363 5.692 27.557 1.00 9.56 O ATOM 3442 N SER B 85 5.646 5.064 26.239 1.00 8.08 N ATOM 3444 CA SER B 85 6.395 5.370 25.021 1.00 11.21 C ATOM 3446 CB SER B 85 6.078 4.253 23.981 1.00 11.13 C ATOM 3449 OG SER B 85 6.861 3.227 24.572 1.00 16.57 O ATOM 3451 C SER B 85 5.939 6.686 24.380 1.00 11.05 C ATOM 3452 O SER B 85 6.348 7.024 23.288 1.00 12.31 O ATOM 3453 N ASP B 86 5.041 7.414 25.025 1.00 12.04 N ATOM 3455 CA ASP B 86 4.571 8.722 24.546 1.00 12.16 C ATOM 3457 CB ASP B 86 3.114 8.874 24.995 1.00 14.37 C ATOM 3460 CG ASP B 86 2.139 7.826 24.420 1.00 14.78 C ATOM 3461 OD1 ASP B 86 2.352 7.224 23.339 1.00 13.22 O ATOM 3462 OD2 ASP B 86 0.980 7.698 24.922 1.00 14.32 O ATOM 3463 C ASP B 86 5.378 9.959 25.004 1.00 12.30 C ATOM 3464 O ASP B 86 5.651 10.258 26.176 1.00 8.45 O ATOM 3465 N ARG B 87 5.768 10.782 24.048 1.00 9.98 N ATOM 3467 CA ARG B 87 6.458 11.956 24.434 1.00 11.52 C ATOM 3469 CB ARG B 87 7.965 11.817 24.224 1.00 10.41 C ATOM 3472 CG ARG B 87 8.570 10.637 24.919 1.00 10.56 C ATOM 3475 CD ARG B 87 8.766 10.836 26.460 1.00 11.53 C ATOM 3478 NE ARG B 87 9.441 9.647 26.971 1.00 15.77 N ATOM 3480 CZ ARG B 87 8.853 8.594 27.512 1.00 15.46 C ATOM 3481 NH1 ARG B 87 7.551 8.577 27.527 1.00 14.29 N ATOM 3484 NH2 ARG B 87 9.539 7.514 27.917 1.00 16.26 N ATOM 3487 C ARG B 87 6.017 13.076 23.494 1.00 12.84 C ATOM 3488 O ARG B 87 5.953 12.916 22.285 1.00 10.06 O ATOM 3489 N ILE B 88 5.998 14.277 24.058 1.00 11.60 N ATOM 3491 CA ILE B 88 5.895 15.516 23.290 1.00 19.61 C ATOM 3493 CB ILE B 88 4.870 16.429 24.022 1.00 12.82 C ATOM 3495 CG1 ILE B 88 4.509 17.594 23.128 1.00 15.21 C ATOM 3498 CD1 ILE B 88 3.329 18.393 23.515 1.00 17.41 C ATOM 3502 CG2 ILE B 88 5.283 16.835 25.342 1.00 13.80 C ATOM 3506 C ILE B 88 7.320 16.076 23.152 1.00 10.33 C ATOM 3507 O ILE B 88 8.086 16.084 24.108 1.00 10.89 O ATOM 3508 N LEU B 89 7.641 16.574 21.972 1.00 7.50 N ATOM 3510 CA LEU B 89 8.899 17.219 21.665 1.00 10.76 C ATOM 3512 CB LEU B 89 9.582 16.550 20.473 1.00 11.08 C ATOM 3515 CG LEU B 89 10.371 15.310 20.923 1.00 15.57 C ATOM 3517 CD1 LEU B 89 9.513 14.253 21.509 1.00 16.83 C ATOM 3521 CD2 LEU B 89 11.614 15.587 21.738 1.00 14.08 C ATOM 3525 C LEU B 89 8.605 18.664 21.313 1.00 10.88 C ATOM 3526 O LEU B 89 7.825 18.955 20.448 1.00 14.16 O ATOM 3527 N TYR B 90 9.384 19.561 21.879 1.00 12.66 N ATOM 3529 CA TYR B 90 9.184 20.975 21.689 1.00 11.30 C ATOM 3531 CB TYR B 90 8.419 21.593 22.845 1.00 7.36 C ATOM 3534 CG TYR B 90 8.855 21.506 24.237 1.00 8.59 C ATOM 3535 CD1 TYR B 90 8.753 20.356 24.947 1.00 11.37 C ATOM 3537 CE1 TYR B 90 9.142 20.289 26.252 1.00 10.53 C ATOM 3539 CZ TYR B 90 9.662 21.424 26.890 1.00 11.48 C ATOM 3540 OH TYR B 90 10.100 21.305 28.179 1.00 12.87 O ATOM 3542 CE2 TYR B 90 9.917 22.587 26.157 1.00 10.68 C ATOM 3544 CD2 TYR B 90 9.482 22.600 24.856 1.00 11.87 C ATOM 3546 C TYR B 90 10.506 21.669 21.549 1.00 9.65 C ATOM 3547 O TYR B 90 11.449 21.401 22.255 1.00 10.81 O ATOM 3548 N SER B 91 10.542 22.570 20.573 1.00 9.62 N ATOM 3550 CA SER B 91 11.779 23.283 20.183 1.00 9.56 C ATOM 3552 CB SER B 91 11.885 23.361 18.667 1.00 7.45 C ATOM 3555 OG SER B 91 10.990 24.322 18.077 1.00 11.78 O ATOM 3557 C SER B 91 11.825 24.653 20.865 1.00 9.77 C ATOM 3558 O SER B 91 10.806 25.180 21.252 1.00 10.48 O ATOM 3559 N SER B 92 12.985 25.262 20.857 1.00 9.65 N ATOM 3561 CA SER B 92 13.204 26.591 21.398 1.00 12.16 C ATOM 3563 CB SER B 92 14.709 26.982 21.486 1.00 13.12 C ATOM 3566 OG SER B 92 15.548 26.350 20.538 1.00 14.59 O ATOM 3568 C SER B 92 12.576 27.644 20.544 1.00 11.62 C ATOM 3569 O SER B 92 12.301 28.677 21.116 1.00 16.48 O ATOM 3570 N ASP B 93 12.147 27.359 19.324 1.00 12.20 N ATOM 3572 CA ASP B 93 11.293 28.188 18.525 1.00 12.09 C ATOM 3574 CB ASP B 93 11.975 28.503 17.199 1.00 14.08 C ATOM 3577 CG ASP B 93 12.197 27.261 16.309 1.00 16.23 C ATOM 3578 OD1 ASP B 93 12.367 26.109 16.806 1.00 12.90 O ATOM 3579 OD2 ASP B 93 12.125 27.398 15.051 1.00 17.42 O ATOM 3580 C ASP B 93 9.817 27.694 18.452 1.00 12.38 C ATOM 3581 O ASP B 93 8.979 28.180 17.657 1.00 14.03 O ATOM 3582 N TRP B 94 9.488 26.774 19.347 1.00 10.13 N ATOM 3584 CA TRP B 94 8.143 26.320 19.634 1.00 11.78 C ATOM 3586 CB TRP B 94 7.312 27.516 20.139 1.00 10.64 C ATOM 3589 CG TRP B 94 7.947 28.062 21.403 1.00 9.63 C ATOM 3590 CD1 TRP B 94 8.580 29.206 21.514 1.00 10.05 C ATOM 3592 NE1 TRP B 94 9.136 29.332 22.769 1.00 10.83 N ATOM 3594 CE2 TRP B 94 8.827 28.239 23.524 1.00 12.03 C ATOM 3595 CD2 TRP B 94 8.153 27.354 22.674 1.00 11.33 C ATOM 3596 CE3 TRP B 94 7.634 26.176 23.225 1.00 12.54 C ATOM 3598 CZ3 TRP B 94 7.860 25.884 24.533 1.00 11.99 C ATOM 3600 CH2 TRP B 94 8.672 26.741 25.311 1.00 13.56 C ATOM 3602 CZ2 TRP B 94 9.146 27.922 24.840 1.00 13.00 C ATOM 3604 C TRP B 94 7.386 25.540 18.572 1.00 12.51 C ATOM 3605 O TRP B 94 6.141 25.744 18.387 1.00 12.94 O ATOM 3606 N LEU B 95 8.149 24.826 17.774 1.00 10.29 N ATOM 3608 CA LEU B 95 7.617 23.705 17.007 1.00 10.18 C ATOM 3610 CB LEU B 95 8.689 23.029 16.168 1.00 9.75 C ATOM 3613 CG LEU B 95 9.283 23.906 15.153 1.00 12.77 C ATOM 3615 CD1 LEU B 95 10.356 23.156 14.444 1.00 14.04 C ATOM 3619 CD2 LEU B 95 8.234 24.220 14.093 1.00 15.77 C ATOM 3623 C LEU B 95 7.230 22.636 18.046 1.00 11.53 C ATOM 3624 O LEU B 95 7.882 22.490 19.086 1.00 9.60 O ATOM 3625 N ILE B 96 6.152 21.909 17.786 1.00 11.73 N ATOM 3627 CA ILE B 96 5.683 20.822 18.656 1.00 7.10 C ATOM 3629 CB ILE B 96 4.329 21.102 19.314 1.00 10.09 C ATOM 3631 CG1 ILE B 96 4.196 22.430 20.048 1.00 10.16 C ATOM 3634 CD1 ILE B 96 5.269 22.658 21.147 1.00 13.15 C ATOM 3638 CG2 ILE B 96 4.062 19.932 20.259 1.00 10.19 C ATOM 3642 C ILE B 96 5.520 19.563 17.766 1.00 11.41 C ATOM 3643 O ILE B 96 4.788 19.544 16.705 1.00 10.52 O ATOM 3644 N TYR B 97 6.191 18.500 18.209 1.00 9.29 N ATOM 3646 CA TYR B 97 6.092 17.186 17.649 1.00 10.84 C ATOM 3648 CB TYR B 97 7.387 16.767 17.041 1.00 11.78 C ATOM 3651 CG TYR B 97 7.812 17.398 15.692 1.00 8.80 C ATOM 3652 CD1 TYR B 97 8.072 18.744 15.580 1.00 7.50 C ATOM 3654 CE1 TYR B 97 8.426 19.267 14.297 1.00 10.82 C ATOM 3656 CZ TYR B 97 8.496 18.470 13.186 1.00 9.25 C ATOM 3657 OH TYR B 97 8.777 19.025 11.997 1.00 11.39 O ATOM 3659 CE2 TYR B 97 8.176 17.146 13.256 1.00 8.97 C ATOM 3661 CD2 TYR B 97 7.856 16.633 14.508 1.00 11.39 C ATOM 3663 C TYR B 97 5.699 16.185 18.783 1.00 11.17 C ATOM 3664 O TYR B 97 5.774 16.472 20.038 1.00 11.86 O ATOM 3665 N LYS B 98 5.252 15.038 18.298 1.00 9.43 N ATOM 3667 CA LYS B 98 4.912 13.885 19.152 1.00 8.78 C ATOM 3669 CB LYS B 98 3.388 13.706 19.208 1.00 8.94 C ATOM 3672 CG LYS B 98 2.805 13.227 17.865 1.00 8.95 C ATOM 3675 CD LYS B 98 1.316 12.827 17.890 1.00 11.21 C ATOM 3678 CE LYS B 98 0.869 11.736 18.861 1.00 9.75 C ATOM 3681 NZ LYS B 98 −0.569 11.508 18.832 1.00 10.33 N ATOM 3685 C LYS B 98 5.599 12.617 18.681 1.00 10.84 C ATOM 3686 O LYS B 98 5.933 12.460 17.487 1.00 11.21 O ATOM 3687 N THR B 99 5.838 11.708 19.621 1.00 10.22 N ATOM 3689 CA THR B 99 6.150 10.334 19.318 1.00 9.66 C ATOM 3691 CB THR B 99 7.710 10.123 19.463 1.00 12.67 C ATOM 3693 OG1 THR B 99 8.050 8.733 19.259 1.00 14.62 O ATOM 3695 CG2 THR B 99 8.189 10.437 20.914 1.00 13.45 C ATOM 3699 C THR B 99 5.340 9.493 20.252 1.00 10.54 C ATOM 3700 O THR B 99 5.108 9.906 21.420 1.00 13.14 O ATOM 3701 N THR B 100 4.784 8.413 19.688 1.00 11.05 N ATOM 3703 CA THR B 100 4.122 7.347 20.395 1.00 10.71 C ATOM 3705 CB THR B 100 2.688 7.046 19.838 1.00 12.11 C ATOM 3707 OG1 THR B 100 2.831 6.710 18.481 1.00 10.59 O ATOM 3709 CG2 THR B 100 1.831 8.385 19.771 1.00 9.70 C ATOM 3713 C THR B 100 4.937 6.064 20.345 1.00 12.36 C ATOM 3714 O THR B 100 4.463 5.051 20.882 1.00 9.43 O ATOM 3715 N ASP B 101 6.147 6.064 19.768 1.00 11.48 N ATOM 3717 CA ASP B 101 6.919 4.832 19.728 1.00 8.19 C ATOM 3719 CB ASP B 101 6.931 4.333 18.253 1.00 9.42 C ATOM 3722 CG ASP B 101 7.531 5.287 17.362 1.00 8.26 C ATOM 3723 OD1 ASP B 101 8.060 6.361 17.787 1.00 6.48 O ATOM 3724 OD2 ASP B 101 7.486 5.084 16.155 1.00 10.50 O ATOM 3725 C ASP B 101 8.325 5.020 20.327 1.00 8.62 C ATOM 3726 O ASP B 101 9.314 4.494 19.794 1.00 9.42 O ATOM 3727 N ALA B 102 8.421 5.759 21.427 1.00 9.30 N ATOM 3729 CA ALA B 102 9.684 5.959 22.146 1.00 8.92 C ATOM 3731 CB ALA B 102 10.089 4.648 22.840 1.00 11.73 C ATOM 3735 C ALA B 102 10.765 6.437 21.191 1.00 10.14 C ATOM 3736 O ALA B 102 11.768 5.787 20.910 1.00 8.75 O ATOM 3737 N TYR B 103 10.482 7.555 20.538 1.00 11.44 N ATOM 3739 CA TYR B 103 11.508 8.278 19.720 1.00 11.53 C ATOM 3741 CB TYR B 103 12.783 8.619 20.551 1.00 12.19 C ATOM 3744 CG TYR B 103 12.597 8.999 22.025 1.00 12.00 C ATOM 3745 CD1 TYR B 103 12.282 10.315 22.398 1.00 13.42 C ATOM 3747 CE1 TYR B 103 12.129 10.692 23.733 1.00 10.35 C ATOM 3749 CZ TYR B 103 12.307 9.737 24.714 1.00 12.04 C ATOM 3750 OH TYR B 103 12.173 10.143 26.013 1.00 15.78 O ATOM 3752 CE2 TYR B 103 12.639 8.441 24.401 1.00 12.70 C ATOM 3754 CD2 TYR B 103 12.826 8.081 23.042 1.00 11.77 C ATOM 3756 C TYR B 103 11.972 7.654 18.464 1.00 10.04 C ATOM 3757 O TYR B 103 12.923 8.127 17.850 1.00 12.44 O ATOM 3758 N GLN B 104 11.204 6.715 17.915 1.00 11.02 N ATOM 3760 CA GLN B 104 11.661 6.200 16.655 1.00 11.75 C ATOM 3762 CB GLN B 104 11.260 4.776 16.530 1.00 14.48 C ATOM 3765 CG GLN B 104 11.996 3.789 17.446 1.00 18.18 C ATOM 3768 CD GLN B 104 11.192 2.511 17.191 1.00 21.14 C ATOM 3769 OE1 GLN B 104 11.534 1.628 16.363 1.00 22.36 O ATOM 3770 NE2 GLN B 104 10.010 2.494 17.829 1.00 22.11 N ATOM 3773 C GLN B 104 11.055 6.964 15.486 1.00 12.44 C ATOM 3774 O GLN B 104 11.678 7.042 14.422 1.00 9.27 O ATOM 3775 N THR B 105 9.805 7.402 15.648 1.00 10.73 N ATOM 3777 CA THR B 105 9.231 8.256 14.622 1.00 12.47 C ATOM 3779 CB THR B 105 8.302 7.521 13.660 1.00 12.84 C ATOM 3781 OG1 THR B 105 7.148 7.143 14.391 1.00 9.11 O ATOM 3783 CG2 THR B 105 8.971 6.206 13.051 1.00 13.18 C ATOM 3787 C THR B 105 8.509 9.447 15.241 1.00 13.88 C ATOM 3788 O THR B 105 8.150 9.366 16.412 1.00 10.75 O ATOM 3789 N PHE B 106 8.458 10.577 14.513 1.00 11.94 N ATOM 3791 CA PHE B 106 7.786 11.715 15.142 1.00 13.81 C ATOM 3793 CB PHE B 106 8.804 12.815 15.394 1.00 12.48 C ATOM 3796 CG PHE B 106 9.843 12.441 16.370 1.00 10.42 C ATOM 3797 CD1 PHE B 106 9.657 12.688 17.722 1.00 10.90 C ATOM 3799 CE1 PHE B 106 10.547 12.287 18.640 1.00 12.22 C ATOM 3801 CZ PHE B 106 11.703 11.615 18.228 1.00 12.53 C ATOM 3803 CE2 PHE B 106 11.879 11.316 16.847 1.00 10.73 C ATOM 3805 CD2 PHE B 106 10.956 11.764 15.949 1.00 9.20 C ATOM 3807 C PHE B 106 6.734 12.247 14.183 1.00 12.03 C ATOM 3808 O PHE B 106 6.966 12.165 13.011 1.00 12.66 O ATOM 3809 N THR B 107 5.745 12.961 14.690 1.00 10.03 N ATOM 3811 CA THR B 107 4.715 13.610 13.897 1.00 12.09 C ATOM 3813 CB THR B 107 3.327 12.936 14.011 1.00 10.99 C ATOM 3815 OG1 THR B 107 2.231 13.621 13.261 1.00 13.44 O ATOM 3817 CG2 THR B 107 3.534 11.546 13.696 1.00 6.47 C ATOM 3821 C THR B 107 4.587 15.061 14.349 1.00 10.62 C ATOM 3822 O THR B 107 4.529 15.328 15.516 1.00 11.81 O ATOM 3823 N LYS B 108 4.675 15.981 13.414 1.00 8.19 N ATOM 3825 CA LYS B 108 4.638 17.363 13.780 1.00 6.78 C ATOM 3827 CB LYS B 108 4.980 18.239 12.589 1.00 6.97 C ATOM 3830 CG LYS B 108 4.758 19.786 12.770 1.00 8.64 C ATOM 3833 CD LYS B 108 5.346 20.499 11.468 1.00 10.24 C ATOM 3836 CE LYS B 108 5.516 21.949 11.654 1.00 9.99 C ATOM 3839 NZ LYS B 108 6.376 22.605 10.559 1.00 7.56 N ATOM 3843 C LYS B 108 3.185 17.602 14.082 1.00 6.60 C ATOM 3844 O LYS B 108 2.340 17.285 13.216 1.00 6.51 O ATOM 3845 N ILE B 109 2.891 18.177 15.252 1.00 7.61 N ATOM 3847 CA ILE B 109 1.503 18.514 15.573 1.00 8.50 C ATOM 3849 CB ILE B 109 1.015 17.742 16.849 1.00 9.03 C ATOM 3851 CG1 ILE B 109 1.990 18.037 18.017 1.00 8.97 C ATOM 3854 CD1 ILE B 109 1.318 17.512 19.330 1.00 7.70 C ATOM 3858 CG2 ILE B 109 1.045 16.280 16.573 1.00 8.27 C ATOM 3862 C ILE B 109 1.235 20.014 15.852 1.00 9.66 C ATOM 3863 O ILE B 109 0.088 20.353 16.058 1.00 11.91 O ATOM 3864 N ARG B 110 2.253 20.866 15.820 1.00 8.83 N ATOM 3866 CA ARG B 110 2.081 22.304 15.741 1.00 10.67 C ATOM 3868 CB ARG B 110 2.001 23.106 17.075 1.00 9.40 C ATOM 3871 CG ARG B 110 1.019 22.512 18.056 1.00 10.12 C ATOM 3874 CD ARG B 110 −0.428 22.908 17.699 1.00 10.77 C ATOM 3877 NE ARG B 110 −1.329 22.540 18.795 1.00 11.62 N ATOM 3879 CZ ARG B 110 −1.885 21.356 19.014 1.00 10.50 C ATOM 3880 NH1 ARG B 110 −1.591 20.279 18.255 1.00 11.81 N ATOM 3883 NH2 ARG B 110 −2.671 21.248 20.061 1.00 13.24 N ATOM 3886 C ARG B 110 3.156 22.952 14.909 1.00 11.39 C ATOM 3887 O ARG B 110 4.293 22.627 15.119 1.00 9.56 O ATOM 3888 N SER B 111 2.761 23.904 14.047 1.00 17.56 N ATOM 3890 CA SER B 111 3.817 24.837 13.596 1.00 18.65 C ATOM 3892 CB SER B 111 3.127 25.831 12.616 1.00 22.08 C ATOM 3895 OG SER B 111 2.761 25.073 11.470 1.00 26.67 O ATOM 3897 C SER B 111 4.330 25.682 14.763 1.00 19.94 C ATOM 3898 O SER B 111 3.671 25.834 15.796 1.00 15.06 O ATOM 3899 N SER B 112 5.413 26.431 14.518 1.00 16.03 N ATOM 3901 CA SER B 112 5.894 27.372 15.454 1.00 16.28 C ATOM 3903 CB SER B 112 7.111 28.164 14.987 1.00 17.02 C ATOM 3906 OG SER B 112 7.529 29.153 15.859 1.00 20.34 O ATOM 3908 C SER B 112 4.787 28.364 15.753 1.00 16.25 C ATOM 3909 O SER B 112 4.290 28.999 14.870 1.00 15.95 O ATOM 3910 N SER B 113 4.640 28.635 17.041 1.00 16.88 N ATOM 3912 CA SER B 113 3.731 29.682 17.470 1.00 17.97 C ATOM 3914 CB SER B 113 3.003 29.096 18.670 1.00 17.19 C ATOM 3917 OG SER B 113 3.823 28.684 19.766 1.00 16.21 O ATOM 3919 C SER B 113 4.457 30.939 17.914 1.00 17.85 C ATOM 3920 O SER B 113 3.821 31.813 18.516 1.00 14.91 O ATOM 3921 N MET B 114 5.771 30.898 17.685 1.00 16.90 N ATOM 3923 CA MET B 114 6.546 31.955 18.261 1.00 19.52 C ATOM 3925 CB MET B 114 8.034 31.609 18.110 1.00 19.35 C ATOM 3928 CG MET B 114 8.865 32.525 18.911 1.00 20.21 C ATOM 3931 SD MET B 114 10.507 31.849 19.144 1.00 22.34 S ATOM 3932 CE MET B 114 11.231 33.131 19.753 1.00 22.35 C ATOM 3936 C MET B 114 6.240 33.293 17.655 1.00 14.52 C ATOM 3937 O MET B 114 6.246 33.425 16.397 1.00 17.95 O ATOM 3938 N GLY B 115 5.853 34.241 18.487 1.00 12.58 N ATOM 3940 CA GLY B 115 5.367 35.478 17.896 1.00 10.36 C ATOM 3943 C GLY B 115 3.889 35.607 17.733 1.00 10.90 C ATOM 3944 O GLY B 115 3.455 36.659 17.290 1.00 9.54 O ATOM 3945 N VAL B 116 3.106 34.578 18.070 1.00 8.88 N ATOM 3947 CA VAL B 116 1.685 34.684 17.836 1.00 6.16 C ATOM 3949 CB VAL B 116 1.192 33.347 17.222 1.00 8.93 C ATOM 3951 CG1 VAL B 116 −0.277 33.381 16.954 1.00 12.38 C ATOM 3955 CG2 VAL B 116 1.831 33.118 15.887 1.00 9.12 C ATOM 3959 C VAL B 116 1.084 34.836 19.201 1.00 8.87 C ATOM 3960 O VAL B 116 1.393 34.087 20.135 1.00 6.19 O ATOM 3961 N CYS B 117 0.053 35.687 19.290 1.00 10.78 N ATOM 3963 CA CYS B 117 −0.424 36.085 20.603 1.00 9.89 C ATOM 3965 CB CYS B 117 −0.726 37.600 20.576 1.00 11.75 C ATOM 3968 SG CYS B 117 −1.669 38.165 22.037 1.00 10.46 S ATOM 3969 C CYS B 117 −1.676 35.307 21.025 1.00 11.58 C ATOM 3970 O CYS B 117 −2.592 35.281 20.309 1.00 11.90 O ATOM 3971 N PRO B 118 −1.677 34.581 22.146 1.00 12.58 N ATOM 3972 CA PRO B 118 −2.804 33.780 22.592 1.00 13.53 C ATOM 3974 CB PRO B 118 −2.317 33.153 23.909 1.00 13.68 C ATOM 3977 CG PRO B 118 −0.851 33.125 23.783 1.00 13.54 C ATOM 3980 CD PRO B 118 −0.466 34.285 22.923 1.00 12.24 C ATOM 3983 C PRO B 118 −3.925 34.758 23.014 1.00 15.56 C ATOM 3984 O PRO B 118 −3.691 35.809 23.599 1.00 11.88 O ATOM 3985 N LYS B 119 −5.144 34.341 22.695 1.00 15.70 N ATOM 3987 CA LYS B 119 −6.297 35.135 23.045 1.00 17.01 C ATOM 3989 CB LYS B 119 −7.383 34.887 22.004 1.00 22.58 C ATOM 3992 CG LYS B 119 −7.327 36.025 20.935 1.00 25.28 C ATOM 3995 CD LYS B 119 −7.787 35.752 19.474 1.00 28.00 C ATOM 3998 CE LYS B 119 −7.749 37.101 18.625 1.00 27.82 C ATOM 4001 NZ LYS B 119 −9.270 37.120 18.547 1.00 33.30 N ATOM 4005 C LYS B 119 −6.695 34.843 24.483 1.00 14.78 C ATOM 4006 O LYS B 119 −7.488 33.952 24.745 1.00 14.72 O ATOM 4007 N ILE B 120 −6.139 35.660 25.388 1.00 13.92 N ATOM 4009 CA ILE B 120 −6.426 35.657 26.829 1.00 13.11 C ATOM 4011 CB ILE B 120 −5.480 34.905 27.638 1.00 13.03 C ATOM 4013 CG1 ILE B 120 −5.391 33.465 27.084 1.00 13.72 C ATOM 4016 CD1 ILE B 120 −4.475 32.514 27.909 1.00 15.32 C ATOM 4020 CG2 ILE B 120 −5.836 35.055 29.195 1.00 13.02 C ATOM 4024 C ILE B 120 −6.460 37.114 27.256 1.00 14.74 C ATOM 4025 O ILE B 120 −5.460 37.769 27.127 1.00 13.53 O ATOM 4026 N ILE B 121 −7.643 37.640 27.540 1.00 10.64 N ATOM 4028 CA LEU B 121 −7.707 39.052 27.854 1.00 12.46 C ATOM 4030 CB LEU B 121 −9.146 39.417 28.156 1.00 12.34 C ATOM 4033 CG LEU B 121 −9.636 40.815 28.565 1.00 12.97 C ATOM 4035 CD1 LEU B 121 −9.438 41.591 27.278 1.00 13.00 C ATOM 4039 CD2 LEU B 121 −11.102 40.869 28.910 1.00 14.52 C ATOM 4043 C LEU B 121 −6.882 39.419 29.071 1.00 11.66 C ATOM 4044 O LEU B 121 −7.171 38.974 30.186 1.00 17.63 O ATOM 4045 N LYS B 122 −6.036 40.414 28.943 1.00 12.56 N ATOM 4047 CA LYS B 122 −5.174 40.807 30.076 1.00 12.21 C ATOM 4049 CB LYS B 122 −3.875 39.996 29.854 1.00 14.60 C ATOM 4052 CG LYS B 122 −2.683 40.335 30.676 1.00 18.10 C ATOM 4055 CD LYS B 122 −3.084 40.179 32.127 1.00 20.56 C ATOM 4058 CE LYS B 122 −1.789 40.064 32.976 1.00 23.92 C ATOM 4061 NZ LYS B 122 −2.311 39.634 34.336 1.00 28.08 N ATOM 4065 C LYS B 122 −4.898 42.321 29.881 1.00 11.03 C ATOM 4066 O LYS B 122 −4.457 42.765 28.795 1.00 12.51 O ATOM 4067 N LYS B 123 −4.977 43.071 30.962 1.00 9.09 N ATOM 4069 CA LYS B 123 −4.695 44.494 30.960 1.00 10.54 C ATOM 4071 CB LYS B 123 −5.098 45.144 32.316 1.00 13.28 C ATOM 4074 CG LYS B 123 −6.591 45.411 32.338 1.00 18.06 C ATOM 4077 CD LYS B 123 −7.163 46.077 33.657 1.00 19.84 C ATOM 4080 CE LYS B 123 −8.710 46.448 33.621 1.00 21.27 C ATOM 4083 NZ LYS B 123 −9.164 47.071 34.941 1.00 22.45 N ATOM 4087 C LYS B 123 −3.165 44.612 30.948 1.00 11.75 C ATOM 4088 O LYS B 123 −2.444 43.790 31.522 1.00 11.87 O ATOM 4089 N CYS B 124 −2.678 45.719 30.408 1.00 10.18 N ATOM 4091 CA CYS B 124 −1.269 45.987 30.396 1.00 8.52 C ATOM 4093 CB CYS B 124 −0.601 45.310 29.211 1.00 9.48 C ATOM 4096 SG CYS B 124 −1.433 45.749 27.654 1.00 10.30 S ATOM 4097 C CYS B 124 −0.982 47.449 30.306 1.00 10.61 C ATOM 4098 O CYS B 124 −1.829 48.242 29.905 1.00 9.26 O ATOM 4099 N ARG B 125 0.284 47.748 30.607 1.00 11.40 N ATOM 4101 CA ARG B 125 0.849 49.061 30.435 1.00 11.65 C ATOM 4103 CB ARG B 125 1.347 49.611 31.780 1.00 15.50 C ATOM 4106 CG ARG B 125 0.223 50.109 32.599 1.00 19.98 C ATOM 4109 CD ARG B 125 0.813 50.748 33.851 1.00 24.00 C ATOM 4112 NE ARG B 125 0.168 51.936 34.448 1.00 31.02 N ATOM 4114 CZ ARG B 125 0.243 52.222 35.802 1.00 33.95 C ATOM 4115 NH1 ARG B 125 0.932 51.414 36.632 1.00 35.25 N ATOM 4118 NH2 ARG B 125 −0.328 53.293 36.385 1.00 34.45 N ATOM 4121 C ARG B 125 1.984 48.994 29.427 1.00 9.67 C ATOM 4122 O ARG B 125 2.250 49.965 28.726 1.00 10.41 O ATOM 4123 N ARG B 126 2.688 47.872 29.345 1.00 7.73 N ATOM 4125 CA ARG B 126 3.725 47.692 28.332 1.00 9.47 C ATOM 4127 CB ARG B 126 5.117 48.052 29.000 1.00 12.34 C ATOM 4130 CG ARG B 126 5.383 47.318 30.299 1.00 14.67 C ATOM 4133 CD ARG B 126 6.628 47.869 31.019 1.00 15.55 C ATOM 4136 NE ARG B 126 7.789 47.510 30.169 1.00 18.94 N ATOM 4138 CZ ARG B 126 9.025 48.071 30.290 1.00 23.10 C ATOM 4139 NH1 ARG B 126 9.273 48.985 31.232 1.00 21.13 N ATOM 4142 NH2 ARG B 126 10.058 47.720 29.495 1.00 24.42 N ATOM 4145 C ARG B 126 3.745 46.218 27.935 1.00 9.85 C ATOM 4146 O ARG B 126 3.088 45.331 28.499 1.00 11.71 O ATOM 4147 N ASP B 127 4.535 45.914 26.928 1.00 8.60 N ATOM 4149 CA ASP B 127 4.497 44.553 26.371 1.00 9.89 C ATOM 4151 CB ASP B 127 5.457 44.441 25.194 1.00 9.11 C ATOM 4154 CG ASP B 127 5.114 45.420 24.054 1.00 11.38 C ATOM 4155 OD1 ASP B 127 4.013 46.027 24.051 1.00 7.79 O ATOM 4156 OD2 ASP B 127 5.990 45.632 23.156 1.00 9.54 O ATOM 4157 C ASP B 127 4.814 43.402 27.338 1.00 8.70 C ATOM 4158 O ASP B 127 4.234 42.332 27.204 1.00 6.87 O ATOM 4159 N SER B 128 5.820 43.613 28.182 1.00 7.27 N ATOM 4161 CA SER B 128 6.115 42.632 29.156 1.00 7.62 C ATOM 4163 CB SER B 128 7.436 42.945 29.874 1.00 6.31 C ATOM 4166 OG SER B 128 7.355 44.167 30.597 1.00 5.17 O ATOM 4168 C SER B 128 5.031 42.266 30.191 1.00 7.45 C ATOM 4169 O SER B 128 5.215 41.316 30.948 1.00 9.76 O ATOM 4170 N ASP B 129 3.906 42.945 30.227 1.00 6.64 N ATOM 4172 CA ASP B 129 2.845 42.526 31.076 1.00 5.36 C ATOM 4174 CB ASP B 129 1.875 43.651 31.314 1.00 6.34 C ATOM 4177 CG ASP B 129 2.481 44.812 32.036 1.00 6.51 C ATOM 4178 OD1 ASP B 129 2.069 45.984 31.794 1.00 8.78 O ATOM 4179 OD2 ASP B 129 3.267 44.674 32.939 1.00 6.56 O ATOM 4180 C ASP B 129 2.032 41.489 30.333 1.00 6.90 C ATOM 4181 O ASP B 129 1.201 40.847 30.961 1.00 9.35 O ATOM 4182 N CYS B 130 2.361 41.241 29.064 1.00 8.72 N ATOM 4184 CA CYS B 130 1.662 40.266 28.283 1.00 11.71 C ATOM 4186 CB CYS B 130 1.347 40.888 26.943 1.00 9.14 C ATOM 4189 SG CYS B 130 0.439 42.509 26.939 1.00 9.10 S ATOM 4190 C CYS B 130 2.368 38.973 28.024 1.00 10.38 C ATOM 4191 O CYS B 130 3.571 38.914 28.083 1.00 14.55 O ATOM 4192 N LEU B 131 1.610 37.961 27.661 1.00 11.47 N ATOM 4194 CA LEU B 131 2.153 36.691 27.206 1.00 12.37 C ATOM 4196 CB LEU B 131 1.016 35.770 26.768 1.00 15.31 C ATOM 4199 CG LEU B 131 0.146 35.345 27.951 1.00 16.72 C ATOM 4201 CD1 LEU B 131 −1.022 34.671 27.445 1.00 15.47 C ATOM 4205 CD2 LEU B 131 0.869 34.400 28.946 1.00 19.03 C ATOM 4209 C LEU B 131 3.075 36.867 25.979 1.00 10.63 C ATOM 4210 O LEU B 131 3.076 37.898 25.337 1.00 9.90 O ATOM 4211 N ALA B 132 4.034 35.970 25.865 1.00 11.14 N ATOM 4213 CA ALA B 132 4.919 35.829 24.726 1.00 8.75 C ATOM 4215 CB ALA B 132 5.620 34.574 24.801 1.00 10.07 C ATOM 4219 C ALA B 132 4.084 35.899 23.441 1.00 9.76 C ATOM 4220 O ALA B 132 2.971 35.444 23.362 1.00 8.80 O ATOM 4221 N GLY B 133 4.458 36.741 22.506 1.00 9.65 N ATOM 4223 CA GLY B 133 3.751 36.937 21.247 1.00 10.68 C ATOM 4226 C GLY B 133 2.702 38.018 21.306 1.00 12.09 C ATOM 4227 O GLY B 133 2.216 38.349 20.251 1.00 9.81 O ATOM 4228 N CYS B 134 2.450 38.606 22.488 1.00 12.02 N ATOM 4230 CA CYS B 134 1.546 39.680 22.637 1.00 11.04 C ATOM 4232 CB CYS B 134 0.570 39.306 23.730 1.00 12.03 C ATOM 4235 SG CYS B 134 −0.369 37.779 23.515 1.00 12.63 S ATOM 4236 C CYS B 134 2.277 40.964 23.014 1.00 9.59 C ATOM 4237 O CYS B 134 3.365 41.040 23.589 1.00 7.88 O ATOM 4238 N VAL B 135 1.627 42.040 22.604 1.00 9.26 N ATOM 4240 CA VAL B 135 2.034 43.417 22.845 1.00 8.42 C ATOM 4242 CB VAL B 135 2.512 44.137 21.572 1.00 9.70 C ATOM 4244 CG1 VAL B 135 3.695 43.379 21.044 1.00 7.35 C ATOM 4248 CG2 VAL B 135 1.409 44.224 20.498 1.00 10.12 C ATOM 4252 C VAL B 135 0.863 44.238 23.405 1.00 10.16 C ATOM 4253 O VAL B 135 −0.243 43.826 23.365 1.00 10.36 O ATOM 4254 N CYS B 136 1.193 45.237 24.198 1.00 9.32 N ATOM 4256 CA CYS B 136 0.230 46.046 24.823 1.00 8.81 C ATOM 4258 CB CYS B 136 0.855 46.732 26.021 1.00 9.87 C ATOM 4261 SG CYS B 136 −0.426 47.442 27.058 1.00 9.28 S ATOM 4262 C CYS B 136 −0.286 47.049 23.783 1.00 6.94 C ATOM 4263 O CYS B 136 0.375 47.951 23.242 1.00 8.86 O ATOM 4264 N GLY B 137 −1.528 46.918 23.504 1.00 6.75 N ATOM 4266 CA GLY B 137 −2.147 47.852 22.595 1.00 9.92 C ATOM 4269 C GLY B 137 −2.508 49.229 23.168 1.00 10.96 C ATOM 4270 O GLY B 137 −2.470 49.438 24.371 1.00 9.72 O ATOM 4271 N PRO B 138 −2.984 50.143 22.297 1.00 11.07 N ATOM 4272 CA PRO B 138 −3.165 51.514 22.722 1.00 10.23 C ATOM 4274 CB PRO B 138 −3.533 52.322 21.450 1.00 12.09 C ATOM 4277 CG PRO B 138 −3.656 51.346 20.402 1.00 12.93 C ATOM 4280 CD PRO B 138 −3.292 49.940 20.879 1.00 10.42 C ATOM 4283 C PRO B 138 −4.269 51.565 23.733 1.00 10.00 C ATOM 4284 O PRO B 138 −4.374 52.585 24.375 1.00 8.95 O ATOM 4285 N ASN B 139 −5.100 50.528 23.821 1.00 11.01 N ATOM 4287 CA ASN B 139 −6.229 50.453 24.757 1.00 11.73 C ATOM 4289 CB ASN B 139 −7.398 49.648 24.143 1.00 12.66 C ATOM 4292 CG ASN B 139 −7.043 48.164 23.935 1.00 15.97 C ATOM 4293 OD1 ASN B 139 −5.850 47.792 23.628 1.00 17.55 O ATOM 4294 ND2 ASN B 139 −8.102 47.349 23.816 1.00 16.59 N ATOM 4297 C ASN B 139 −5.851 49.894 26.113 1.00 11.34 C ATOM 4298 O ASN B 139 −6.692 49.722 26.961 1.00 11.56 O ATOM 4299 N GLY B 140 −4.580 49.571 26.321 1.00 11.16 N ATOM 4301 CA GLY B 140 −4.135 49.044 27.588 1.00 11.37 C ATOM 4304 C GLY B 140 −4.515 47.571 27.751 1.00 13.70 C ATOM 4305 O GLY B 140 −4.560 47.091 28.894 1.00 11.23 O ATOM 4306 N PHE B 141 −4.740 46.853 26.652 1.00 10.89 N ATOM 4308 CA PHE B 141 −4.891 45.423 26.705 1.00 12.78 C ATOM 4310 CB PHE B 141 −6.306 44.957 26.389 1.00 10.06 C ATOM 4313 CG PHE B 141 −7.336 45.362 27.436 1.00 11.78 C ATOM 4314 CD1 PHE B 141 −7.711 44.504 28.471 1.00 7.36 C ATOM 4316 CE1 PHE B 141 −8.597 44.947 29.489 1.00 9.72 C ATOM 4318 CZ PHE B 141 −9.128 46.180 29.446 1.00 12.47 C ATOM 4320 CE2 PHE B 141 −8.703 47.083 28.426 1.00 12.66 C ATOM 4322 CD2 PHE B 141 −7.866 46.658 27.409 1.00 11.40 C ATOM 4324 C PHE B 141 −3.889 44.781 25.793 1.00 9.78 C ATOM 4325 O PHE B 141 −3.560 45.224 24.695 1.00 12.17 O ATOM 4326 N CYS B 142 −3.707 43.526 26.091 1.00 7.08 N ATOM 4328 CA CYS B 142 −2.768 42.694 25.376 1.00 9.17 C ATOM 4330 CB CYS B 142 −2.189 41.557 26.226 1.00 9.73 C ATOM 4333 SG CYS B 142 −1.323 42.018 27.751 1.00 10.02 S ATOM 4334 C CYS B 142 −3.320 42.140 24.095 1.00 9.02 C ATOM 4335 O CYS B 142 −4.431 41.691 24.074 1.00 11.66 O ATOM 4336 N GLY B 143 −2.562 42.134 23.011 1.00 9.08 N ATOM 4338 CA GLY B 143 −3.060 41.566 21.754 1.00 10.12 C ATOM 4341 C GLY B 143 −2.022 41.372 20.679 1.00 10.66 C ATOM 4342 O GLY B 143 −0.853 41.535 21.057 1.00 6.24 O ATOM 4343 N SER B 144 −2.373 40.993 19.468 1.00 10.91 N ATOM 4345 CA SER B 144 −1.348 40.819 18.471 1.00 12.34 C ATOM 4347 CB SER B 144 −1.704 40.101 17.211 1.00 14.54 C ATOM 4350 OG SER B 144 −2.592 39.027 17.539 1.00 17.92 O ATOM 4352 C SER B 144 −0.582 42.056 18.066 1.00 14.23 C ATOM 4353 O SER B 144 −1.210 43.081 18.067 1.00 14.50 O ATOM 4354 OXT SER B 144 0.614 42.013 17.708 1.00 13.17 O ATOM 4355 S SO4 X 1 −8.456 59.328 25.375 1.00 62.73 S ATOM 4356 O1 SO4 X 1 −7.367 58.486 24.804 1.00 61.80 O ATOM 4357 O2 SO4 X 1 −8.232 60.230 26.492 1.00 59.94 O ATOM 4358 O3 SO4 X 1 −8.605 60.235 24.188 1.00 60.50 O ATOM 4359 O4 SO4 X 1 −9.327 58.362 26.132 1.00 58.47 O ATOM 4360 S SO4 X 2 21.150 27.268 10.030 1.00 44.73 S ATOM 4361 O1 SO4 X 2 22.620 27.325 10.129 1.00 45.48 O ATOM 4362 O2 SO4 X 2 20.443 28.463 10.581 1.00 44.20 O ATOM 4363 O3 SO4 X 2 20.634 27.169 8.666 1.00 44.34 O ATOM 4364 O4 SO4 X 2 20.873 25.984 10.659 1.00 44.14 O ATOM 4365 S SO4 X 3 11.477 23.840 10.225 1.00 54.03 S ATOM 4366 O1 SO4 X 3 12.992 23.747 10.387 1.00 52.64 O ATOM 4367 O2 SO4 X 3 10.500 24.718 10.950 1.00 52.07 O ATOM 4368 O3 SO4 X 3 11.312 24.727 9.071 1.00 55.72 O ATOM 4369 O4 SO4 X 3 11.011 22.642 9.534 1.00 52.59 O ATOM 4370 S SO4 X 4 15.596 8.604 28.993 1.00 75.74 S ATOM 4371 O1 SO4 X 4 16.683 9.569 29.222 1.00 76.36 O ATOM 4372 O2 SO4 X 4 14.997 8.662 30.328 1.00 75.37 O ATOM 4373 O3 SO4 X 4 14.478 8.820 28.043 1.00 74.08 O ATOM 4374 O4 SO4 X 4 16.378 7.433 28.550 1.00 75.08 O ATOM 4375 O5 PG4 X 100 12.072 43.213 7.525 1.00 39.50 O ATOM 4377 C8 PG4 X 100 12.548 43.607 8.885 1.00 37.25 C ATOM 4380 C7 PG4 X 100 11.817 43.689 10.251 1.00 33.81 C ATOM 4383 O4 PG4 X 100 11.369 44.773 11.185 1.00 34.32 O ATOM 4384 C6 PG4 X 100 9.889 44.956 11.391 1.00 24.12 C ATOM 4387 C5 PG4 X 100 8.581 45.829 11.434 1.00 26.64 C ATOM 4390 O3 PG4 X 100 7.471 45.386 10.455 1.00 29.66 O ATOM 4391 C4 PG4 X 100 6.503 46.017 9.644 1.00 28.44 C ATOM 4394 C3 PG4 X 100 5.903 45.321 8.386 1.00 30.07 C ATOM 4397 O2 PG4 X 100 6.866 44.799 7.455 1.00 30.65 O ATOM 4398 C2 PG4 X 100 6.761 43.940 6.323 1.00 32.24 C ATOM 4401 C1 PG4 X 100 8.049 43.362 5.699 1.00 35.20 C ATOM 4404 O1 PG4 X 100 8.996 44.184 4.980 1.00 36.32 O ATOM 4406 O5 PG4 X 101 −8.880 40.691 20.549 1.00 42.26 O ATOM 4408 C8 PG4 X 101 −9.332 39.861 19.445 1.00 37.50 C ATOM 4411 C7 PG4 X 101 −10.060 40.099 18.098 1.00 36.74 C ATOM 4414 O4 PG4 X 101 −9.381 39.225 17.139 1.00 35.25 O ATOM 4415 C6 PG4 X 101 −9.588 38.588 15.865 1.00 37.24 C ATOM 4418 C5 PG4 X 101 −8.334 38.332 15.007 1.00 38.38 C ATOM 4421 O3 PG4 X 101 −7.427 37.351 15.506 1.00 40.25 O ATOM 4422 C4 PG4 X 101 −6.369 36.662 14.847 1.00 42.96 C ATOM 4425 C3 PG4 X 101 −5.678 35.666 15.803 1.00 44.49 C ATOM 4428 O2 PG4 X 101 −4.238 35.808 16.035 1.00 47.79 O ATOM 4429 C2 PG4 X 101 −3.655 35.990 17.344 1.00 47.22 C ATOM 4432 C1 PG4 X 101 −4.649 36.586 18.393 1.00 46.24 C ATOM 4435 O1 PG4 X 101 −4.356 37.565 19.404 1.00 39.61 O ATOM 4437 O HOH W 1 0.000 58.790 14.540 0.50 11.72 O ATOM 4440 O HOH W 2 0.000 24.853 14.540 0.50 12.82 O ATOM 4443 O HOH W 3 5.569 8.132 30.455 1.00 18.15 O ATOM 4446 O HOH W 4 4.308 30.482 21.389 1.00 18.25 O ATOM 4449 O HOH W 5 10.470 13.439 29.078 1.00 19.96 O ATOM 4452 O HOH W 6 10.334 71.418 15.207 1.00 20.56 O ATOM 4455 O HOH W 7 12.261 35.986 13.669 1.00 17.17 O ATOM 4458 O HOH W 8 8.715 36.629 25.110 1.00 18.41 O ATOM 4461 O HOH W 9 −2.731 12.411 24.763 1.00 16.82 O ATOM 4464 O HOH W 10 9.048 44.966 26.900 1.00 27.01 O ATOM 4467 O HOH W 11 6.415 10.293 33.357 1.00 20.43 O ATOM 4470 O HOH W 12 3.956 33.537 27.379 1.00 19.80 O ATOM 4473 O HOH W 13 1.940 39.628 17.828 1.00 22.19 O ATOM 4476 O HOH W 14 −1.947 16.381 18.388 1.00 15.41 O ATOM 4479 O HOH W 15 2.281 12.528 31.170 1.00 16.86 O ATOM 4482 O HOH W 16 13.356 11.260 29.109 1.00 30.83 O ATOM 4485 O HOH W 17 13.419 59.715 28.278 1.00 20.40 O ATOM 4488 O HOH W 18 9.109 68.562 12.889 1.00 19.53 O ATOM 4491 O HOH W 19 20.514 43.163 16.705 1.00 35.54 O ATOM 4494 O HOH W 20 11.132 68.532 31.061 1.00 21.92 O ATOM 4497 O HOH W 21 10.592 50.742 22.338 1.00 19.02 O ATOM 4500 O HOH W 22 5.729 58.346 14.919 1.00 20.60 O ATOM 4503 O HOH W 23 6.395 47.767 25.855 1.00 21.74 O ATOM 4506 O HOH W 24 −5.301 15.063 18.514 1.00 19.43 O ATOM 4509 O HOH W 25 8.102 51.134 19.350 1.00 19.33 O ATOM 4512 O HOH W 26 5.711 21.642 8.152 1.00 20.85 O ATOM 4515 O HOH W 27 14.942 71.340 21.781 1.00 18.33 O ATOM 4518 O HOH W 28 −7.820 61.894 18.143 1.00 22.35 O ATOM 4521 O HOH W 29 12.150 15.157 30.288 1.00 23.42 O ATOM 4524 O HOH W 30 15.645 73.649 26.582 1.00 24.21 O ATOM 4527 O HOH W 31 27.619 71.256 33.365 1.00 28.16 O ATOM 4530 O HOH W 32 11.082 30.951 10.399 1.00 27.41 O ATOM 4533 O HOH W 33 5.396 8.177 16.702 1.00 19.60 O ATOM 4536 O HOH W 34 5.838 58.094 28.696 1.00 27.76 O ATOM 4539 O HOH W 35 −4.247 15.097 22.657 1.00 19.58 O ATOM 4542 O HOH W 36 4.961 50.326 20.161 1.00 15.13 O ATOM 4545 O HOH W 37 2.553 43.907 16.824 1.00 19.84 O ATOM 4548 O HOH W 38 3.560 44.117 10.280 1.00 26.18 O ATOM 4551 O HOH W 39 10.369 70.392 29.020 1.00 23.61 O ATOM 4554 O HOH W 40 −4.440 61.433 32.419 1.00 32.08 O ATOM 4557 O HOH W 41 5.516 52.935 20.837 1.00 15.86 O ATOM 4560 O HOH W 42 −5.375 40.911 18.995 1.00 24.57 O ATOM 4563 O HOH W 43 5.490 39.538 12.342 1.00 31.58 O ATOM 4566 O HOH W 44 7.558 35.834 20.807 1.00 19.05 O ATOM 4569 O HOH W 45 4.729 60.236 29.586 1.00 15.57 O ATOM 4572 O HOH W 46 2.279 32.794 23.898 1.00 18.78 O ATOM 4575 O HOH W 47 7.077 42.538 11.012 1.00 18.63 O ATOM 4578 O HOH W 48 −1.052 10.367 16.178 1.00 28.44 O ATOM 4581 O HOH W 49 1.958 57.209 15.560 1.00 22.98 O ATOM 4584 O HOH W 50 18.613 78.830 29.537 1.00 23.72 O ATOM 4587 O HOH W 51 3.528 15.368 44.050 1.00 29.21 O ATOM 4590 O HOH W 52 −6.051 41.809 33.733 1.00 28.67 O ATOM 4593 O HOH W 53 1.499 14.231 35.574 1.00 21.04 O ATOM 4596 O HOH W 54 −4.121 67.336 15.091 1.00 22.73 O ATOM 4599 O HOH W 55 −0.324 37.396 17.017 1.00 24.78 O ATOM 4602 O HOH W 56 9.434 33.285 26.515 1.00 29.61 O ATOM 4605 O HOH W 57 18.143 33.757 12.186 1.00 29.20 O ATOM 4608 O HOH W 58 13.968 43.052 13.635 1.00 19.33 O ATOM 4611 O HOH W 59 12.408 28.178 11.443 1.00 26.19 O ATOM 4614 O HOH W 60 18.506 19.558 21.258 1.00 24.26 O ATOM 4617 O HOH W 61 18.217 58.246 23.895 1.00 26.84 O ATOM 4620 O HOH W 62 −1.232 38.434 27.190 1.00 22.02 O ATOM 4623 O HOH W 63 4.995 69.915 14.221 1.00 20.73 O ATOM 4626 O HOH W 64 5.965 33.589 21.250 1.00 16.38 O ATOM 4629 O HOH W 65 5.460 68.539 10.698 1.00 22.04 O ATOM 4632 O HOH W 66 3.600 33.068 21.336 1.00 20.02 O ATOM 4635 O HOH W 67 19.422 69.743 22.196 1.00 20.58 O ATOM 4638 O HOH W 68 −1.767 73.135 14.951 1.00 36.16 O ATOM 4641 O HOH W 69 1.423 10.955 37.687 1.00 24.08 O ATOM 4644 O HOH W 70 14.952 66.300 14.549 1.00 25.64 O ATOM 4647 O HOH W 71 8.446 76.529 30.667 1.00 28.89 O ATOM 4650 O HOH W 72 11.897 46.124 15.353 1.00 19.96 O ATOM 4653 O HOH W 73 13.452 67.576 36.982 1.00 24.13 O ATOM 4656 O HOH W 74 6.475 25.912 11.779 1.00 23.93 O ATOM 4659 O HOH W 75 0.016 23.977 35.410 1.00 34.89 O ATOM 4662 O HOH W 76 8.360 44.168 23.192 1.00 21.03 O ATOM 4665 O HOH W 77 6.253 52.072 17.601 1.00 18.19 O ATOM 4668 O HOH W 78 10.791 23.588 29.362 1.00 29.93 O ATOM 4671 O HOH W 79 −0.729 47.967 15.636 1.00 24.15 O ATOM 4674 O HOH W 80 −5.946 41.064 26.119 1.00 25.29 O ATOM 4677 O HOH W 81 12.859 22.724 30.930 1.00 26.81 O ATOM 4680 O HOH W 82 12.877 23.410 23.827 1.00 21.68 O ATOM 4683 O HOH W 83 −0.579 45.550 17.331 1.00 19.14 O ATOM 4686 O HOH W 84 12.717 45.547 17.915 1.00 18.82 O ATOM 4689 O HOH W 85 4.653 54.216 29.700 1.00 27.98 O ATOM 4692 O HOH W 86 8.433 5.156 37.077 1.00 22.40 O ATOM 4695 O HOH W 87 −6.017 69.464 20.826 1.00 39.33 O ATOM 4698 O HOH W 88 2.994 48.232 22.818 1.00 15.43 O ATOM 4701 O HOH W 89 9.112 75.208 34.001 1.00 37.26 O ATOM 4704 O HOH W 90 −0.072 73.879 18.702 1.00 33.81 O ATOM 4707 O HOH W 91 −3.833 23.229 27.552 1.00 26.34 O ATOM 4710 O HOH W 92 8.797 34.594 22.914 1.00 19.91 O ATOM 4713 O HOH W 93 −5.093 38.811 23.433 1.00 26.55 O ATOM 4716 O HOH W 94 3.630 46.545 13.630 1.00 20.19 O ATOM 4719 O HOH W 95 12.956 75.740 24.756 1.00 17.89 O ATOM 4722 O HOH W 96 −2.191 46.712 19.298 1.00 19.11 O ATOM 4725 O HOH W 97 0.254 26.340 16.863 1.00 28.81 O ATOM 4728 O HOH W 98 18.526 19.241 16.363 1.00 26.18 O ATOM 4731 O HOH W 99 −1.153 25.253 20.015 1.00 25.87 O ATOM 4734 O HOH W 100 18.488 22.025 16.032 1.00 33.45 O ATOM 4737 O HOH W 101 17.029 78.337 21.107 1.00 24.35 O ATOM 4740 O HOH W 102 9.100 17.423 9.981 1.00 25.36 O ATOM 4743 O HOH W 103 13.927 80.205 30.359 1.00 32.23 O ATOM 4746 O HOH W 104 16.562 30.601 12.293 1.00 31.46 O ATOM 4749 O HOH W 105 0.233 75.590 20.523 1.00 22.05 O ATOM 4752 O HOH W 106 5.911 74.458 14.901 1.00 34.42 O ATOM 4755 O HOH W 107 −3.053 9.843 23.060 1.00 33.67 O ATOM 4758 O HOH W 108 13.624 60.717 14.932 1.00 22.75 O ATOM 4761 O HOH W 109 −11.385 35.553 17.771 1.00 25.06 O ATOM 4764 O HOH W 110 9.197 52.364 11.557 1.00 33.98 O ATOM 4767 O HOH W 111 15.172 64.468 37.267 1.00 34.40 O ATOM 4770 O HOH W 112 3.808 9.889 15.981 1.00 32.38 O ATOM 4773 O HOH W 113 3.113 38.346 15.162 1.00 28.61 O ATOM 4776 O HOH W 114 10.293 33.312 10.568 1.00 36.43 O ATOM 4779 O HOH W 115 17.095 48.164 10.932 1.00 31.46 O ATOM 4782 O HOH W 116 0.976 52.541 28.901 1.00 28.51 O ATOM 4785 O HOH W 117 −3.666 54.865 23.887 1.00 34.22 O ATOM 4788 O HOH W 118 16.943 16.288 34.339 1.00 25.43 O ATOM 4791 O HOH W 119 −10.149 64.450 29.311 1.00 32.72 O ATOM 4794 O HOH W 120 19.689 59.775 21.082 1.00 26.63 O ATOM 4797 O HOH W 121 −3.691 48.425 17.743 1.00 19.17 O ATOM 4800 O HOH W 122 21.430 73.034 23.048 1.00 28.42 O ATOM 4803 O HOH W 123 10.265 27.752 12.773 1.00 31.81 O ATOM 4806 O HOH W 124 0.806 63.954 34.184 1.00 23.12 O ATOM 4809 O HOH W 125 12.287 7.157 27.671 1.00 24.45 O ATOM 4812 O HOH W 126 18.944 29.156 12.709 1.00 26.63 O ATOM 4815 O HOH W 127 −7.243 66.210 21.853 1.00 29.41 O ATOM 4818 O HOH W 128 −0.149 12.004 35.548 1.00 27.14 O ATOM 4821 O HOH W 129 4.382 24.565 9.438 1.00 28.77 O ATOM 4824 O HOH W 130 −3.342 25.747 27.914 1.00 29.86 O ATOM 4827 O HOH W 131 −1.529 50.070 17.418 1.00 22.20 O ATOM 4830 O HOH W 132 14.650 72.436 15.638 1.00 25.05 O ATOM 4833 O HOH W 133 17.878 62.716 31.362 1.00 25.14 O ATOM 4836 O HOH W 134 9.227 79.817 20.659 1.00 29.29 O ATOM 4839 O HOH W 135 −3.314 37.852 25.395 1.00 23.68 O ATOM 4842 O HOH W 136 0.000 73.500 14.540 0.50 41.45 O ATOM 4845 O HOH W 137 4.068 51.191 26.923 1.00 23.45 O ATOM 4848 O HOH W 138 20.008 71.988 20.726 1.00 27.68 O ATOM 4851 O HOH W 139 13.234 5.847 32.263 1.00 26.27 O ATOM 4854 O HOH W 140 21.749 63.563 28.203 1.00 39.60 O ATOM 4857 O HOH W 141 −5.465 73.144 23.338 1.00 32.11 O ATOM 4860 O HOH W 142 6.574 53.636 15.141 1.00 33.57 O ATOM 4863 O HOH W 143 −4.638 57.202 19.896 1.00 36.81 O ATOM 4866 O HOH W 144 23.482 65.349 22.878 1.00 40.79 O ATOM 4869 O HOH W 145 0.000 36.784 14.540 0.50 36.06 O ATOM 4872 O HOH W 146 11.619 25.587 27.445 1.00 38.30 O ATOM 4875 O HOH W 147 3.678 0.181 34.918 1.00 35.60 O ATOM 4878 O HOH W 148 −0.922 55.118 30.320 1.00 34.56 O ATOM 4881 O HOH W 149 10.578 21.194 36.599 1.00 24.21 O ATOM 4884 O HOH W 150 6.844 79.063 19.415 1.00 27.87 O ATOM 4887 O HOH W 151 6.947 33.486 28.230 1.00 35.70 O ATOM 4890 O HOH W 152 9.923 10.379 12.113 1.00 24.52 O ATOM 4893 O HOH W 153 16.798 8.264 42.844 1.00 32.24 O ATOM 4896 O HOH W 154 17.274 19.428 9.768 1.00 27.77 O ATOM 4899 O HOH W 155 8.826 21.865 11.163 1.00 26.29 O ATOM 4902 O HOH W 156 27.143 68.891 26.526 1.00 36.72 O ATOM 4905 O HOH W 157 16.246 79.665 31.172 1.00 29.11 O ATOM 4908 O HOH W 158 11.534 25.541 24.515 1.00 26.28 O ATOM 4911 O HOH W 159 10.685 45.664 4.374 1.00 37.97 O ATOM 4914 O HOH W 160 −5.673 61.783 21.945 1.00 24.53 O ATOM 4917 O HOH W 161 22.240 79.362 26.912 1.00 29.20 O ATOM 4920 O HOH W 162 0.803 5.435 33.148 1.00 26.13 O ATOM 4923 O HOH W 163 2.950 3.896 25.431 1.00 32.10 O ATOM 4926 O HOH W 164 8.803 73.744 14.259 1.00 34.41 O ATOM 4929 O HOH W 165 18.289 18.636 29.961 1.00 28.63 O ATOM 4932 O HOH W 166 −5.968 58.836 17.376 1.00 37.46 O ATOM 4935 O HOH W 167 10.900 60.135 29.293 1.00 33.46 O ATOM 4938 O HOH W 168 15.619 63.656 29.877 1.00 29.28 O ATOM 4941 O HOH W 169 19.391 35.752 15.989 1.00 33.30 O ATOM 4944 O HOH W 170 4.391 49.703 24.737 1.00 29.63 O ATOM 4947 O HOH W 171 −3.594 71.525 30.335 1.00 41.73 O ATOM 4950 O HOH W 172 9.494 60.912 37.398 1.00 38.36 O ATOM 4953 O HOH W 173 −6.047 39.285 21.011 1.00 43.07 O ATOM 4956 O HOH W 174 3.638 25.697 34.715 1.00 27.03 O ATOM 4959 O HOH W 175 8.238 39.535 27.775 1.00 20.24 O ATOM 4962 O HOH W 176 9.002 24.171 31.532 1.00 19.88 O ATOM 4965 O HOH W 177 −1.497 9.498 20.373 1.00 28.11 O ATOM 4968 O HOH W 178 6.540 52.061 12.149 1.00 28.41 O ATOM 4971 O HOH W 179 17.795 51.254 16.536 1.00 28.05 O ATOM 4974 O HOH W 180 23.668 63.439 29.730 1.00 34.47 O ATOM 4977 O HOH W 181 13.724 58.071 15.406 1.00 28.65 O ATOM 4980 O HOH W 182 1.468 63.179 36.696 1.00 37.17 O ATOM 4983 O HOH W 183 −4.503 17.545 28.757 1.00 31.69 O ATOM 4986 O HOH W 184 1.190 54.806 14.848 1.00 40.39 O ATOM 4989 O HOH W 185 −5.451 48.707 30.980 1.00 47.15 O ATOM 4992 O HOH W 186 26.237 64.077 25.969 1.00 34.48 O ATOM 4995 O HOH W 187 4.495 59.364 32.171 1.00 47.06 O ATOM 4998 O HOH W 188 18.371 17.436 12.446 1.00 31.73 O ATOM 5001 O HOH W 189 −4.452 13.028 19.551 1.00 42.23 O ATOM 5004 O HOH W 190 6.392 20.193 39.348 1.00 34.18 O ATOM 5007 O HOH W 191 11.097 47.282 24.689 1.00 27.20 O ATOM 5010 O HOH W 192 6.759 70.006 33.832 1.00 30.32 O ATOM 5013 O HOH W 193 −5.313 55.974 21.971 1.00 37.30 O ATOM 5016 O HOH W 194 11.186 33.502 23.735 1.00 31.59 O ATOM 5019 O HOH W 195 5.132 39.962 25.444 1.00 26.14 O ATOM 5022 O HOH W 196 15.843 61.406 28.650 1.00 37.75 O ATOM 5025 O HOH W 197 6.121 49.560 11.474 1.00 19.12 O ATOM 5028 O HOH W 198 −3.691 64.262 32.648 1.00 33.48 O ATOM 5031 O HOH W 199 11.259 30.201 26.404 1.00 39.04 O ATOM 5034 O HOH W 200 15.467 10.721 25.734 1.00 30.17 O ATOM 5037 O HOH W 201 17.074 37.421 26.045 1.00 35.86 O ATOM 5040 O HOH W 202 11.016 31.027 23.765 1.00 36.02 O ATOM 5043 O HOH W 203 −7.480 39.258 24.530 1.00 29.90 O ATOM 5046 O HOH W 204 20.829 20.177 22.398 1.00 31.08 O ATOM 5049 O HOH W 205 12.637 77.832 32.808 1.00 35.88 O ATOM 5052 O HOH W 206 0.194 9.694 40.528 1.00 37.33 O ATOM 5055 O HOH W 207 −0.188 5.179 28.683 1.00 39.12 O ATOM 5058 O HOH W 208 5.619 72.852 32.734 1.00 30.88 O ATOM 5061 O HOH W 209 10.039 20.268 34.085 1.00 23.56 O ATOM 5064 O HOH W 210 4.491 1.662 26.042 1.00 36.43 O ATOM 5067 O HOH W 211 9.543 34.268 32.189 1.00 44.91 O ATOM 5070 O HOH W 212 18.162 14.065 30.634 1.00 40.62 O ATOM 5073 O HOH W 213 19.262 76.397 39.166 1.00 33.47 O ATOM 5076 O HOH W 214 6.323 57.214 33.232 1.00 31.54 O ATOM 5079 O HOH W 215 21.017 16.074 15.221 1.00 46.56 O ATOM 5082 O HOH W 216 11.669 67.591 11.375 1.00 42.53 O ATOM 5085 O HOH W 217 11.940 61.510 12.504 1.00 33.55 O ATOM 5088 O HOH W 218 9.102 63.809 9.568 1.00 39.79 O ATOM 5091 O HOH W 219 18.537 12.895 24.532 1.00 33.67 O ATOM 5094 O HOH W 220 −3.399 32.781 31.853 1.00 42.62 O ATOM 5097 O HOH W 221 19.656 30.684 9.101 1.00 30.56 O ATOM 5100 O HOH W 222 −7.746 31.604 23.654 1.00 51.51 O ATOM 5103 O HOH W 223 6.410 70.578 12.271 1.00 43.62 O ATOM 5106 O HOH W 224 18.414 26.816 21.935 1.00 33.30 O ATOM 5109 O HOH W 225 −4.562 73.658 18.557 1.00 40.27 O ATOM 5112 O HOH W 226 1.834 45.214 14.792 1.00 33.35 O ATOM 5115 O HOH W 227 −6.456 16.978 25.284 1.00 42.58 O ATOM 5118 O HOH W 228 1.485 47.197 34.415 1.00 43.14 O ATOM 5121 O HOH W 229 8.518 58.008 29.660 1.00 46.30 O ATOM 5124 O HOH W 230 13.320 17.345 43.256 1.00 36.98 O ATOM 5127 O HOH W 231 −2.219 27.551 21.949 1.00 36.33 O ATOM 5130 O HOH W 232 20.763 34.788 14.546 1.00 43.67 O ATOM 5133 O HOH W 233 25.392 78.634 37.424 1.00 50.95 O ATOM 5136 O HOH W 234 29.860 76.534 27.279 1.00 28.22 O ATOM 5139 O HOH W 235 8.669 29.399 11.804 1.00 41.52 O ATOM 5142 O HOH W 236 18.163 30.488 20.723 1.00 54.55 O ATOM 5145 O HOH W 237 26.581 79.924 33.310 1.00 41.69 O ATOM 5148 O HOH W 238 −7.320 17.258 21.238 1.00 34.51 O ATOM 5151 O HOH W 239 15.141 41.048 27.454 1.00 28.78 O ATOM 5154 O HOH W 240 15.351 24.118 24.643 1.00 36.57 O ATOM 5157 O HOH W 241 10.464 50.247 25.346 1.00 40.45 O ATOM 5160 O HOH W 242 −7.555 20.139 21.792 1.00 33.87 O ATOM 5163 O HOH W 243 −3.365 10.200 32.114 1.00 37.80 O ATOM 5166 O HOH W 244 7.111 14.218 46.259 1.00 37.31 O ATOM 5169 O HOH W 245 −0.497 7.203 22.295 1.00 35.75 O ATOM 5172 O HOH W 246 5.000 4.810 39.815 1.00 26.98 O ATOM 5175 O HOH W 247 7.136 49.342 8.646 1.00 26.65 O ATOM 5178 O HOH W 248 −5.792 22.456 25.116 1.00 32.36 O ATOM 5181 O HOH W 249 16.203 12.016 16.320 1.00 31.39 O ATOM 5184 O HOH W 250 −4.880 16.340 30.695 1.00 42.96 O ATOM 5187 O HOH W 251 5.949 42.046 22.980 1.00 36.30 O ATOM 5190 O HOH W 252 0.573 29.971 34.228 1.00 47.16 O ATOM 5193 O HOH W 253 −0.987 29.373 20.834 1.00 36.44 O ATOM 5196 O HOH W 254 19.444 19.199 18.718 1.00 37.77 O ATOM 5199 O HOH W 255 24.736 80.148 25.329 1.00 39.85 O ATOM 5202 O HOH W 256 11.691 61.111 31.739 1.00 32.31 O ATOM 5205 O HOH W 257 2.864 3.875 17.924 1.00 38.15 O ATOM 5208 O HOH W 258 14.511 16.067 9.859 1.00 49.07 O ATOM 5211 O HOH W 259 6.041 15.719 44.019 1.00 38.12 O ATOM 5214 O HOH W 260 −3.500 11.317 28.973 1.00 31.78 O ATOM 5217 O HOH W 261 20.113 18.209 14.238 1.00 39.21 O ATOM 5220 O HOH W 262 12.901 53.061 26.970 1.00 43.64 O ATOM 5223 O HOH W 263 13.748 78.590 19.665 1.00 45.08 O ATOM 5226 O HOH W 264 −5.789 32.286 21.059 1.00 36.44 O ATOM 5229 O HOH W 265 −2.186 52.836 16.940 1.00 30.43 O ATOM 5232 O HOH W 266 7.623 9.946 10.664 1.00 39.43 O ATOM 5235 O HOH W 267 12.879 65.624 39.396 1.00 46.93 O ATOM 5238 O HOH W 268 3.762 3.939 41.903 1.00 46.14 O ATOM 5241 O HOH W 269 17.828 31.637 16.048 1.00 47.28 O ATOM 5244 O HOH W 270 3.542 70.692 35.072 1.00 36.22 O ATOM 5247 O HOH W 271 9.189 65.423 36.649 1.00 36.55 O ATOM 5250 O HOH W 272 18.089 19.294 34.947 1.00 32.14 O ATOM 5253 O HOH W 273 12.998 3.363 21.479 1.00 27.18 O ATOM 5256 O HOH W 274 0.719 28.723 15.348 1.00 38.43 O ATOM 5259 O HOH W 275 15.569 18.739 42.772 1.00 42.03 O ATOM 5262 O HOH W 276 −3.457 55.073 30.607 1.00 45.58 O ATOM 5265 O HOH W 277 5.522 31.715 14.294 1.00 32.09 O ATOM 5268 O HOH W 278 −2.657 35.531 31.044 1.00 34.38 O ATOM 5271 O HOH W 279 12.105 63.958 11.282 1.00 37.18 O ATOM 5274 O HOH W 280 18.747 47.105 16.449 1.00 34.97 O ATOM 5277 O HOH W 281 5.977 55.901 30.172 1.00 38.23 O ATOM 5280 O HOH W 282 25.844 78.740 23.059 1.00 40.40 O ATOM 5283 O HOH W 283 15.278 9.678 17.509 1.00 38.76 O ATOM 5286 O HOH W 284 21.498 72.061 18.923 1.00 37.11 O ATOM 5289 O HOH W 285 18.724 50.881 14.447 1.00 44.73 O ATOM 5292 O HOH W 286 −11.218 48.480 24.128 1.00 42.03 O ATOM 5295 O HOH W 287 −1.136 28.910 34.251 1.00 38.99 O ATOM 5298 O HOH W 288 11.120 58.787 33.554 1.00 34.94 O ATOM 5301 O HOH W 289 −10.583 56.470 31.910 1.00 43.90 O ATOM 5304 O HOH W 290 2.255 4.906 22.735 1.00 33.76 O ATOM 5307 O HOH W 291 −4.463 53.493 27.131 1.00 37.65 O ATOM 5310 O HOH W 292 26.026 81.949 31.499 1.00 45.48 O ATOM 5313 O HOH W 293 7.442 76.447 16.369 1.00 41.23 O ATOM 5316 O HOH W 294 −3.686 27.401 26.198 1.00 35.10 O ATOM 5319 O HOH W 295 21.577 22.219 15.316 1.00 41.70 O ATOM 5322 O HOH W 296 −2.025 13.772 19.436 1.00 19.84 O ATOM 5325 O HOH W 297 4.552 56.197 33.298 1.00 45.25 O ATOM 5328 O HOH W 298 14.647 47.199 23.522 1.00 48.24 O ATOM 5331 O HOH W 299 15.390 62.007 13.636 1.00 42.37 O ATOM 5334 O HOH W 300 10.428 26.350 31.879 1.00 42.60 O ATOM 5337 O HOH W 301 14.718 5.189 45.549 1.00 45.54 O ATOM 5340 O HOH W 302 8.946 22.508 33.873 1.00 40.76 O ATOM 5343 O HOH W 303 16.384 27.295 24.851 1.00 41.97 O ATOM 5346 O HOH W 304 3.772 7.760 47.728 1.00 41.52 O ATOM 5349 O HOH W 305 10.504 78.455 18.240 1.00 40.71 O ATOM 5352 O HOH W 306 1.711 18.621 39.993 1.00 46.98 O ATOM 5355 O HOH W 307 −2.232 37.318 29.555 1.00 52.79 O ATOM 5358 O HOH W 308 17.695 84.082 24.846 1.00 35.15 O ATOM 5361 O HOH W 309 9.444 81.936 21.257 1.00 43.21 O ATOM 5364 O HOH W 310 18.962 14.586 41.000 1.00 33.95 O ATOM 5367 O HOH W 311 17.224 59.864 27.056 1.00 44.86 O ATOM 5370 O HOH W 312 8.196 32.729 30.403 1.00 46.43 O ATOM 5373 O HOH W 313 6.716 21.920 35.622 1.00 40.07 O ATOM 5376 O HOH W 314 −8.178 51.601 21.128 1.00 26.96 O ATOM 5379 O HOH W 315 21.626 23.241 10.371 1.00 46.41 O ATOM 5382 O HOH W 316 1.992 8.600 16.619 1.00 47.59 O ATOM 5385 O HOH W 317 −9.110 62.976 22.532 1.00 41.04 O ATOM 5388 O HOH W 318 10.186 78.289 31.815 1.00 35.64 O ATOM 5391 O HOH W 319 7.452 56.107 15.200 1.00 47.41 O ATOM 5394 O HOH W 320 16.416 21.307 39.421 1.00 47.41 O ATOM 5397 O HOH W 321 −0.758 31.033 19.795 1.00 50.41 O ATOM 5400 O HOH W 322 −0.518 16.247 39.461 1.00 34.97 O ATOM 5403 O HOH W 323 −1.833 51.374 29.019 1.00 40.40 O ATOM 5406 O HOH W 324 15.835 54.684 14.540 1.00 41.84 O ATOM 5409 O HOH W 325 2.870 15.482 46.756 1.00 35.83 O ATOM 5412 O HOH W 326 −4.606 53.769 17.686 1.00 34.90 O ATOM 5415 O HOH W 327 16.007 35.046 26.985 1.00 43.92 O ATOM 5418 O HOH W 328 16.481 19.803 36.607 1.00 33.16 O ATOM 5421 O HOH W 329 −3.099 12.154 36.959 1.00 43.78 O ATOM 5424 O HOH W 330 8.836 66.515 9.394 1.00 38.90 O ATOM 5427 O HOH W 331 −5.196 55.555 14.903 1.00 41.11 O ATOM 5430 O HOH W 332 3.673 20.670 43.076 1.00 49.53 O ATOM 5433 O HOH W 333 14.210 57.558 29.654 1.00 38.52 O ATOM 5436 O HOH W 334 −4.440 26.268 30.382 1.00 41.01 O ATOM 5439 O HOH W 335 0.303 5.327 26.203 1.00 40.13 O ATOM 5442 O HOH W 336 23.927 25.223 17.079 1.00 49.07 O ATOM 5445 O HOH W 337 18.154 2.532 36.875 1.00 48.40 O ATOM 5448 O HOH W 338 −1.578 9.589 38.800 1.00 45.45 O ATOM 5451 O HOH W 339 10.755 44.148 30.134 1.00 44.26 O ATOM 5454 O HOH W 340 −5.779 19.722 29.027 1.00 44.44 O ATOM 5457 O HOH W 341 2.605 2.158 29.438 1.00 45.14 O ATOM 5460 O HOH W 342 13.174 27.958 25.103 1.00 47.67 O ATOM 5463 O HOH W 343 2.644 4.022 32.067 1.00 44.27 O ATOM 5466 O HOH W 344 −6.107 51.428 30.348 1.00 37.84 O ATOM 5469 O HOH W 345 8.444 46.539 25.407 1.00 27.68 O ATOM 5472 O HOH W 346 0.216 9.594 15.345 0.50 19.30 O ATOM 5475 O HOH W 347 7.419 52.115 28.750 1.00 43.60 O ATOM 5478 O HOH W 348 1.084 74.672 17.236 1.00 43.61 O ATOM 5481 O HOH W 349 9.086 56.763 13.070 1.00 32.67 O ATOM 5484 O HOH W 350 10.168 36.128 33.057 1.00 46.83 O ATOM 5487 O HOH W 351 6.222 30.908 12.092 1.00 40.82 O ATOM 5490 O HOH W 352 18.711 54.082 21.232 1.00 40.98 O ATOM 5493 O HOH W 353 7.045 24.081 34.543 1.00 40.00 O ATOM 5496 O HOH W 354 19.551 36.122 18.141 1.00 39.80 O ATOM 5499 O HOH W 355 8.323 35.863 26.985 1.00 42.79 O ATOM 5502 O HOH W 356 15.768 76.605 17.198 1.00 41.62 O ATOM 5505 O HOH W 357 7.713 14.211 11.015 1.00 38.48 O ATOM 5508 O HOH W 358 −0.007 19.970 39.379 1.00 40.86 O ATOM 5511 O HOH W 359 4.894 10.472 11.299 1.00 43.58 O ATOM 5514 O HOH W 360 −7.013 19.983 24.789 1.00 41.53 O ATOM 5517 O HOH W 361 11.882 69.046 38.268 1.00 37.09 O ATOM 5520 O HOH W 362 0.847 38.127 31.394 1.00 44.71 O ATOM 5523 O HOH W 363 12.543 77.215 35.944 1.00 46.47 O ATOM 5526 O HOH W 364 −0.275 5.281 20.527 1.00 49.03 O ATOM 5529 O HOH W 365 3.852 47.193 33.824 1.00 45.52 O ATOM 5532 O HOH W 366 5.374 28.678 10.725 1.00 55.58 O ATOM 5535 O HOH W 367 7.148 2.729 15.160 1.00 47.51 O ATOM 5538 O HOH W 368 5.850 −0.340 39.838 1.00 45.55 O ATOM 5541 O HOH W 369 20.182 18.359 34.879 1.00 43.84 O ATOM 5544 O HOH W 370 9.912 3.925 45.795 1.00 45.61 O ATOM 5547 O HOH W 371 −8.844 50.990 27.544 1.00 43.80 O ATOM 5550 O HOH W 372 −8.869 42.344 32.264 1.00 36.72 O ATOM 5553 O HOH W 373 15.800 13.628 27.582 1.00 41.13 O ATOM 5556 O HOH W 374 18.901 14.505 33.218 1.00 41.62 O ATOM 5559 O HOH W 375 1.853 79.517 18.137 1.00 46.62 O ATOM 5562 O HOH W 376 16.236 86.830 33.769 1.00 41.68 O ATOM 5565 O HOH W 377 4.608 27.482 37.133 1.00 45.58 O ATOM 5568 O HOH W 378 −3.757 32.642 19.402 1.00 46.75 O ATOM 5571 O HOH W 379 14.471 32.910 26.576 1.00 53.42 O ATOM 5574 O HOH W 380 −1.037 46.322 34.315 1.00 47.01 O ATOM 5577 O HOH W 381 1.793 32.853 32.560 1.00 42.76 O ATOM 5580 O HOH W 382 −2.051 73.756 29.908 1.00 44.88 O ATOM 5583 O HOH W 383 14.083 81.363 21.948 1.00 41.19 O ATOM 5586 O HOH W 384 17.211 49.125 17.870 1.00 49.94 O ATOM 5589 O HOH W 385 6.061 38.796 30.019 1.00 37.05 O ATOM 5592 O HOH W 386 3.701 38.468 32.113 1.00 49.50 O ATOM 5595 O HOH W 387 11.997 45.720 26.358 1.00 43.58 O ATOM 5598 O HOH W 388 3.891 33.001 30.507 1.00 47.99 O ATOM 5601 O HOH W 389 14.616 70.545 40.365 1.00 45.64 O ATOM 5604 O HOH W 390 14.574 22.055 41.425 1.00 52.12 O ATOM 5607 O HOH W 391 4.346 13.355 48.148 1.00 52.19 O ATOM 5610 O HOH W 392 27.169 76.192 24.082 1.00 39.17 O ATOM 5613 O HOH W 393 9.571 38.797 27.006 1.00 32.47 O ATOM 5616 O HOH W 394 8.508 43.203 25.738 1.00 39.98 O ATOM 5619 O HOH W 395 19.082 54.659 19.221 1.00 40.35 O ATOM 5622 O HOH W 396 20.830 42.893 13.607 1.00 39.95 O ATOM 5625 O HOH W 397 5.319 20.544 43.801 1.00 49.19 O ATOM 5628 O HOH W 398 −0.686 77.185 18.682 1.00 48.70 O ATOM 5631 O HOH W 399 13.037 80.375 31.401 1.00 34.60 O ATOM 5634 O HOH W 400 9.194 62.157 37.713 1.00 52.16 O ATOM 5637 O HOH W 401 −7.980 17.818 43.735 1.00 46.19 O ATOM 5640 O HOH W 402 23.280 74.562 20.442 1.00 52.69 O ATOM 5643 O HOH W 403 14.240 61.150 32.356 1.00 47.70 O ATOM 5646 O HOH W 404 −0.769 78.275 30.180 1.00 50.98 O ATOM 5649 O HOH W 405 −5.626 22.219 29.113 1.00 46.52 O ATOM 5652 O HOH W 406 15.541 64.739 13.428 1.00 46.05 O ATOM 5655 O HOH W 407 7.527 37.758 26.580 1.00 25.51 O ATOM 5658 O HOH W 408 24.624 74.521 22.385 1.00 38.85 O ATOM 5661 O HOH W 409 7.728 50.654 28.168 1.00 47.15 O ATOM 5664 O HOH W 410 15.960 6.626 32.420 1.00 43.54 O ATOM 5667 O HOH W 411 −7.813 53.566 25.122 1.00 52.09 O ATOM 5670 O HOH W 412 7.890 45.778 27.572 1.00 30.54 O ATOM 5673 O HOH W 413 21.095 43.371 18.127 1.00 38.67 O ATOM 5676 O HOH W 414 12.440 22.432 33.478 1.00 44.89 O

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1. Use of a microprotein or a polynucleotide encoding said microprotein for the preparation of a pharmaceutical composition for treating or preventing a disease that can be treated or prevented by inhibiting the activity of tryptase.
 2. The use of claim 1, wherein the microprotein comprises at least six cysteine residues, of which six cysteine residues are connected via disulphide bonds so as to form a cystine knot.
 3. The use of claim 1, wherein the microprotein has a peptide backbone with an open or a circular conformation.
 4. The use of claim 1, wherein the microprotein comprises the amino acid motif CX₃-CX₄-CX₄₋₇-CX₁-CX₄₋₅-CX₅₋₇ (SEQ ID NO: 18), with X meaning independently from each other any amino acid residue.
 5. The use of claim 1, wherein the microprotein has a length of between 28 and 40 amino acids.
 6. The use of claim 1, wherein the microprotein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence depicted in any one of SEQ ID NOs: 1 to 15; (b) the amino acid sequence depicted in SEQ ID NO: 16 or 17; (c) a fragment of the amino acid sequence of (a) or (b), said fragment being capable of inhibiting tryptase activity; and (d) a functional equivalent in which at least one residue of the amino acid sequence or of the fragment of any one of (a) to (c) is substituted, added and/or deleted, said functional equivalent being capable of inhibiting tryptase activity.
 7. The use of claim 1, wherein the microprotein is fused to barnase.
 8. The use of claim 7, wherein the barnase is inactive.
 9. The use of claim 1, wherein the microprotein is administered to the patient in the form of a gene delivery vector which expresses the microprotein.
 10. The use of claim 9, wherein the cells are transformed with the vector ex vivo and the transformed cells are administered to the patient.
 11. The use of claim 1, wherein the disease is selected from the group consisting of asthma, inflammation, psoriasis, pulmonary fibrosis, an interstitial lung disease, rheumatoid arthritis, gingivitis, peridontitis, an allergic reaction, allergic rhinitis, osteoarthritis, atherosclerosis, angiogenesis, multiple sclerosis and cancer.
 12. A method for the treatment of an individual in need of inhibiting the activity of tryptase comprising administering to said individual an effective amount of a pharmaceutical composition comprising the microprotein as defined in claim 1 or a polynucleotide encoding said microprotein and, optionally, a pharmaceutically acceptable carrier.
 13. Use of the microprotein as defined in claim 1 or a polynucleotide encoding said microprotein for inhibiting tryptase activity.
 14. The use of claim 13, which is in vitro.
 15. Use of the microprotein as defined in claim 1 for purifying tryptase.
 16. Use of the microprotein as defined in claim 1 as a carrier molecule for tryptase or a derivative thereof.
 17. Use of the microprotein as defined in claim 1 for detecting and/or quantifying tryptase in a sample.
 18. A method for diagnosing a disorder associated with an aberrant abundance of tryptase in a given cell, tissue, organ or organism, comprising (a) contacting a sample from said cell, tissue, organ or organism with a microprotein as defined in claim 1 under conditions allowing binding between tryptase and the microprotein; (b) determining the amount of the microprotein bound to tryptase; and (c) diagnosing a disorder when the determined amount is above or below a standard amount.
 19. Use of the microprotein as defined in claim 1 or a polynucleotide for diagnosing a disease related to an aberrant expression of tryptase.
 20. A kit comprising a microprotein as defined in claim 1 and a manual for carrying out a method of diagnosing a disease related to an aberrant expression of tryptase and, optionally, means of detection or a standard tryptase sample.
 21. (canceled)
 22. A fusion protein comprising barnase and a microprotein.
 23. The fusion protein of claim 22, wherein said barnase is inactive.
 24. A fusion protein comprising barnase and a microprotein, wherein the microprotein is a microprotein as defined in claim
 1. 25. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of claim
 1. 26. The nucleic acid molecule of claim 25, which is DNA, cDNA, or RNA.
 27. A vector comprising the nucleic acid molecule of claim
 25. 28. The vector of claim 27, wherein the nucleotide sequence encoding the fusion protein is operably linked to one or more expression control sequences allowing the expression of the fusion protein in a host cell.
 29. A host cell genetically engineered with the nucleic acid molecule of claim 25 or a vector comprising said nucleic acid molecule.
 30. A method for preparing a fusion protein comprising cultivating the host cell of claim 29 under conditions that the fusion protein encoded by said nucleic acid molecule or vector is expressed; and recovering the fusion protein from the culture.
 31. The method of claim 30, wherein said recovering comprises a step in which the fusion protein is purified by way of binding the barnase moiety of the fusion protein to barstar.
 32. A fusion protein obtainable by the method of claim
 30. 33. A method of detecting or purifying the fusion protein of claim 21 comprising the step of contacting the fusion protein with barstar.
 34. The method of claim 33, wherein barstar is immobilized.
 35. Use of the fusion protein of claim 21 for producing a crystal of said fusion protein.
 36. A crystal of a microprotein fused with barnase.
 37. The crystal of claim 36, wherein the microprotein is a microprotein.
 38. The crystal of claim 36, wherein the barnase is inactive.
 39. The crystal of claim 36, wherein the microprotein fused with barnase comprises the amino acid sequence set forth in SEQ ID NO:
 19. 40. The crystal of claim 36, which belongs to space group C222₁.
 41. The crystal of claim 36, having the unit cell dimensions of a=73.981 Å, b=217.820 Å and c=58.322 Å, α=β=γ=90°.
 42. The crystal of claim 36 which has the crystal coordinates as depicted in Table
 6. 43. A method of preparing the crystal of claim 36 comprising the steps of: (a) providing an aqueous solution comprising a microprotein fused with barnase; (b) providing a reservoir solution comprising a precipitating agent; (c) mixing a volume of said aqueous solution with a volume of said reservoir solution thereby forming a mixed volume; and (d) crystallizing at least a portion of said mixed volume.
 44. The method of claim 43, wherein the reservoir solution comprises at least one compound selected from the group consisting of HEPES, NaCl, PEG 100, PEG 200, PEG 400, PEG 500, PEG 600, PEG 800, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 5000, PEG 6000, PEG 7000, PEG 8000, ammonium sulphate, ammonium acetate, sodium sulphate, organic solvents, isopropanol, citrate buffer, Tris buffer, cacodylate buffer, MES-buffer, dithiothreitol, octylglycopyranoside and uranylacetate.
 45. The method of claim 43, wherein the reservoir solution comprises PEG
 400. 46. The method of claim 43, wherein the reservoir solution comprises PEG at a concentration between 5 and 10%.
 47. The method of claim 43, wherein the reservoir solution has pH 6.5.
 48. The method of claim 43, wherein said crystallizing is carried out according to the sitting drop method.
 49. A crystal of a microprotein obtainable by the method of claim
 43. 50. A method for determining the three-dimensional structure of a microprotein, comprising the steps of: (a) performing an X-ray diffraction analysis of a crystal of a microprotein fused with barnase; (b) computationally processing the diffraction data obtained in step (a) in order to determine the three-dimensional structure of the crystal components and thereby to obtain crystal coordinates; and (c) storing the obtained crystal coordinates on a data storage medium.
 51. The method of claim 50, wherein step (b) further comprises to use data on the three-dimensional structure of barnase not being fused to the microprotein for modelling the structure of the barnase moiety within the fusion protein.
 52. The method of claim 50, wherein the crystal is a crystal of a microprotein fused with a barnase.
 53. Use of the crystal of claim 36 or structure data obtainable therefrom for designing or identifying a compound as a drug.
 54. The use of claim 53, wherein the drug is capable of inhibiting a serine protease.
 55. The use of claim 54, wherein the serine protease is tryptase.
 56. A machine-readable data storage medium comprising the structure data of the crystal of claim 36 and which, when read by an appropriate machine, can be used to display a three-dimensional representation of the microprotein contained in the crystal or a portion thereof.
 57. The data storage medium of claim 56 obtainable by the method comprising the steps of: (a) performing an X-ray diffraction analysis of a crystal of a microprotein fused with barnase; (b) computationally processing the diffraction data obtained in step (a) in order to determine the three-dimensional structure of the crystal components and thereby to obtain crystal coordinates; and (c) storing the obtained crystal coordinates on a data storage medium.
 58. A method of identifying a compound capable of binding to a microprotein comprising the steps of: (a) performing a fitting reaction by computational means between a microprotein and a candidate compound using the structure data of the microprotein obtainable from the crystal of claim 36 and structure data of said compound; and (b) determining whether the candidate compound is capable of binding the microprotein based on the data obtained in the fitting reaction.
 59. Use of a crystal of a microprotein fused with a barnase or structure data obtainable therefrom or the data storage medium of claim 56 for identifying a compound capable of binding to a microprotein.
 60. A method for optimizing the binding activity of a microprotein to a target molecule comprising the steps of: (a) determining by computational means one or more residues of the microprotein which participate in the binding interaction with the target molecule using structure data of the microprotein obtainable from a crystal of claim 36 and structure data of the target molecule; and (b) modifying in the microprotein one or more of said determined residues so as to optimize the binding activity of the microprotein to the target molecule.
 61. The method of claim 60, wherein the structure data of the microprotein and the target molecule are obtained from a crystallized co-complex of said microprotein and target molecule.
 62. The method of claim 60, wherein the structure data of the microprotein the binding activity of which is to be optimized is obtained by computationally superimposing its structure on the structure data of another microprotein's crystal.
 63. Use of the crystal of claim 35 for optimizing the three-dimensional structure of said microprotein with respect to its binding and/or inhibiting activity to a target molecule.
 64. The method of claim 60, wherein the target molecule is a tryptase.
 65. A pharmaceutical composition comprising the crystal of claim 36 and, optionally, a pharmaceutically acceptable carrier.
 66. Use of a crystal of claim 36 for the preparation of a pharmaceutical composition for treating or preventing a disease that can be treated or prevented by inhibiting the activity of tryptase.
 67. The use of claim 66, wherein the disease is selected from the group consisting of asthma, inflammation, psoriasis, pulmonary fibrosis, an interstitial lung disease, rheumatoid arthritis, gingivitis, peridontitis, an allergic reaction, allergic rhinitis, osteoarthritis, atherosclerosis, angiogenesis, multiple sclerosis or cancer. 